ionworks ht - mdc.custhelp.com

DRAFT October 16, 2004 1:11 pmIonWorksHTtile.fm

IonWorks HT

Users Guide

P/N 5900-0349D

DRAFT October 16, 2004 1:11 pmIonWorksHTtile.fm

Copyright 2002 by Molecular Devices CorporationAll rights reserved. Printed in USA.

This manual contains information protected by copyright. No part of this manual may be photocopied or reproduced in any form without prior written consent from Molecular Devices Corporation.

Trademark Information

IonWorks™ is a registered trademark of Molecular Devices Corporation (“MDC”) and is not to be used in any type of promotion or advertising without permission from MDC.

All other trademarks are the property of their respective owners.

Molecular Devices Corporation Disclaimer

Molecular Devices Corporation (“MDC”) makes no warranties, express or implied, including without limitation the implied warranties of merchantability and fitness for a particular purpose, regarding IonWorks™. MDC does not warrant, guarantee or make any representations regarding the use or the results of the use of IonWorks™ in terms of its correctness, accuracy, reliability, currentness or otherwise, and the entire risk as to the results and the performance of IonWorks™ is assumed by you. The exclusion of implied warranties is not permitted in some states; therefore, the above exclusion may not apply to you.

In no event will MDC, its directors, officers, employees or agents be liable to you for any consequential, incidental or indirect damages (including damages for loss of business profits, business interruption, loss of business information, and the like) arising out of the use or inability to use IonWorks™ even if MDC has been advised of the possibility of such damages. Because some states do not allow the exclusion or limitation of liability for consequential or incidental damages, the above may not apply to you. MDC's liability to you for actual damages from any cause whatsoever, and regardless of the form of the action (whether in contract, tort [including negligence), product liability or otherwise), will be limited to $50.

Contents
1 Introduction
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1In This Chapter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1

Overview of the IonWorks HT System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2How the IonWorks HT System Works . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2IonWorks HT System Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3Operating the IonWorks HT System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3

IonWorks HT System Specifications and Requirements . . . . . . . . . . . . . . . . . . . . . . . 1-5Electrical Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-5Physical Specifications and Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-5Vacuum Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-5

Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6Documentation User Attention Words . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6Chemical Hazard Warning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6Chemical Waste Hazard Warning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6About MSDSs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6Ordering MSDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7Instrument Safety Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7Before Operating the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7

Safety Alert Symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-8Electrical Symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-8Nonelectrical Symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-8

2 System DescriptionOverview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1

In This Chapter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1IonWorks HT System Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2IonWorks HT System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2

System Cabinet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3Cabinet Upper Compartment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3Cabinet Lower Compartment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3Source and Waste Containers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3

Experiment Stage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4Components of the Experiment Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4Electronics-Head Soak-Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-5

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Electronics-Head Wash-Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-5PatchPlate Station with Clamping Frame . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-5Source and Waste Bottles for the PatchPlate Station. . . . . . . . . . . . . . . . . . . . .2-5Fluidics-Head Wash-Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-6Source and Waste Bottles for the F-Head Wash . . . . . . . . . . . . . . . . . . . . . . . .2-6Compound Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-6Cells Suspension Reservoir . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-6Buffer Reservoir . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-6

12-Channel Fluidics Head . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-7Features of the Fluidics Head. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-7Fluidics Head Mechanism of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-7

48-Channel Electronics Head. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-8Features of the Electronics Head . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-8Electronics Head Mechanism of Action—The Amplifier System . . . . . . . . . . . .2-8

PatchPlate Consumable. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-9Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-9Well Arrangement and Capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-9PatchPlate Packaging. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-9

Electronics Cabinet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-10Components of the Electronics Cabinet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-10Power Supply Module. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-10Control Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-10Motor Drive Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-10

Fluidics Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-11Peristaltic Pumps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-11Fluid Control and Networking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-11Plenum and Debubblers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-11Vacuum Regulator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-12

Source and Waste Bottle System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-13Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-13Source Bottles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-13Waste Bottles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-13

System Computer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-15Computer Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-15

3 Starting Up and Shutting Down the SystemOverview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-1

In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-1Starting-Up the IonWorks HT System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-2

About Starting-Up the System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-2Procedure for Start-Up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-2

Shutting-Down the IonWorks HT System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-3

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Procedure for Shutting-Down . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-3

4 System Software DescriptionOverview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-1

In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-1Main Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-2

IonWorks Software Main Window. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-2Main Window with View PatchPlate Selected . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-4

Title Bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-4Status Bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-4

IonWorks Software Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-5Menu Bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-5File Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-5Edit Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-6View Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-6Experiment Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-7Utilities Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-7Window Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-8Data Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-8Help Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-9Toolbar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-9Data File Title Bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-9PatchPlate Area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-10Plate Legend and Statistics Area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-11Configure Filter Metrics Pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-12Configure Analysis Method Pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-13Configure Hits Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-14

Main Window with View Compound Plate Selected . . . . . . . . . . . . . . . . . . . . . . . . . .4-16Compound Plate Area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-16Viewing Compound Plate Well Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-17Viewing the Analysis Metrics Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-18

Dialog Boxes and Windows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-19Becoming Familiar with Dialog Boxes and Windows . . . . . . . . . . . . . . . . . . . .4-19

File Menu Dialog Boxes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-20Open Data Dialog Box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-20Save As Dialog Box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-21Configure Trace Export Dialog Box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-22Configure Metrics Export Dialog Box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-23

Experimental Protocol Properties Dialog Boxes. . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-24About the Protocol Properties Dialog Box . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-24Name File Tab. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-24Start —> Seal Test Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-25

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Access—>End Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-28Waveform Editor Dialog Box. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-31Set Single-Addition Screen Parameters Dialog Box. . . . . . . . . . . . . . . . . . . . .4-34Analyze Data Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-38Design Analysis Metric Dialog Box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-39Edit Metrics Menu Dialog Box. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-42Edit Filter Dialog Box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-43Native Filter Dialog Box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-45

View Menu Dialog Boxes and Windows. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-46View Active Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-46Experiment Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-47Trace Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-48IonWorks Messages Dialog Box. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-51

5 Operating the SystemOverview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-1

In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-1Overview of Running an Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-2

Experiment Operations Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-2Setting Up the IonWorks HT System for an Experimental Run . . . . . . . . . . . . . . . . . .5-3

About the Setting Up the System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-3Preparing the Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-4

Cell Densities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-4Cell Preparation Requirements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-4Window of Opportunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-4

Preparing Buffer Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-5About Buffer Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-5External Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-5Internal Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-5Determining Voltage-Offsets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-5

Preparing the Perforation Agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-7About Perforating Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-7Using Amphotericin B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-7

Preparing Compound Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-8When to Prepare the Compound Plate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-8Compound Plate Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-8Dispensing from Compound Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-8Concentration of Compounds in the Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-8

Preparing the Fluidics System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-9Preparing the Liquid-Delivery System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-9

Preparing the Experiment Stage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-11About the Experiment Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-11

vi IonWorks HT User Guide

Table of Contents

Loading a PatchPlate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-11Preparing the Fluid Lines and 12-Channel Pipettor . . . . . . . . . . . . . . . . . . . . .5-11

Setting-Up the Experiment Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-12Loading and Modifying the Default Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . .5-12

Completing the Experiment Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-14Prepare and Load the Perforation Agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-14Check Solutions and Stations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-14Preparing and Loading the Compound Plates . . . . . . . . . . . . . . . . . . . . . . . . .5-14Preparing and Loading the Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-14Loading a New PatchPlate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-15Starting the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-16Viewing the Experiment Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-16Post-Experiment Operations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-17

Exporting Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-18About Exporting Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-18Exporting Data for a Single Well . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-18Exporting Data for Multiple Wells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-20

Importing a Data File into Microsoft Excel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-21About Importing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-21Importing Data Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-21

Exporting Metrics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-23About Exporting Metrics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-23

Importing a Metrics File into Microsoft Excel® . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-25

About Importing Metrics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-25Importing Metric Data Files. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-25

Shutting Down the System After Completing All Daily Experiments . . . . . . . . . . . . .5-27About Shutting Down . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-27Cleaning the System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-27

A MaintenanceAppendix Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1

In This Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1Daily Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-2

About Daily Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-2Start of Day . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-2Prior to Each Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-2End of the Day . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-2PatchPlate Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-3

Chloriding the E-Head Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-4Purpose of Chloride Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-4Applying a Chloride Coating to the E-Head . . . . . . . . . . . . . . . . . . . . . . . . . . . A-4

Detecting a Salt Bridge. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-6

IonWorks HT User Guide vii

Table of Contents

Purpose of Salt-Bridge Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-6Detecting a Salt Bridge. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-6

Test Pipettor Utility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-7Purpose of Test Pipettor Utility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-7Using the Test Pipettor Utility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-7

B Product Warranty StatementIonWorks HT Product Warranty Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-1

Instrument Warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-1Accessory Warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-1Warranty Exclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-2Remedy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-2Warranty Disclaimer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-2

C Customer SupportContacting Molecular Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-1

Contacting via the Web. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-1Contacting by Telephone, Fax or Email . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-1

D Mapping DiagramsFluidics Head Mapping Diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-1Electronics Head Mapping diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2Compound Plate Mapping Diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-3

Index

viii IonWorks HT User Guide

1
Introduction 1
Overview

In This Chapter This chapter provides an overview of the IonWorks® HT System. This includes the principles of operation, system components, how the system operates and system specifications and requirements. Additionally, there is information regarding user and instrument safety.

Topic See Page

Overview of the IonWorks HT System 1-2

IonWorks HT System Specifications and Requirements 1-5

Safety 1-6

Safety Alert Symbols 1-8

IonWorks HT Users Guide 1-1

Chapter 1 Introduction

Overview of the IonWorks HT System

Introduction The IonWorks® HT system is a high-throughput electrophysiology instrument that performs “whole-cell” voltage-clamp recordings of ion channels. Ion channels are protein molecules that span the lipid bi-layer of the cell membrane. They underlie many physiological processes such as neural signaling, cardiovascular function, immune cell activation and muscle contraction. The gold-standard measurement methodology for studying ion channels is the patch-clamp technique. The reason this technique is so powerful is the fact that you are able to directly measure the fast electrical currents of ion channel activity while simultaneously controlling the cell’s membrane potential. Although very powerful, the patch-clamp technique is extremely labor-intensive and has a limited measurement throughput.

The novel technology behind the IonWorks HT couples the high measurement fidelity obtained in a standard patch-clamp device with the rapid measurement throughput, required for high-throughput screening and drug discovery. The IonWorks HT system uses the PatchPlate™ consumable to provide a custom 384-well platform for obtaining simultaneous, parallel electrical recordings. Integration of a 48-channel electronics head and a 12-channel pipettor equips the IonWorks HT system with electrical-measurement and fluidic-handling capabilities necessary for a full-fledged screening instrument. The technology of the IonWorks HT system enables scientists to design cell-based assays for monitoring the behavior of different types of ion channels, including the voltage-gated ion channels.

How the IonWorksHT System Works

The basic objective of the IonWorks HT system is to perform parallel “whole-cell” electrophysiological measurements. To achieve this, individual cells are positioned on small pores at the interface of two isolated fluid chambers, each containing a separate electrode. Both fluid chambers contain balanced salt solutions that provide the medium for flow of electrical currents. The small pores serve as the only contact point and consequently the only path for the flow of electrical currents between these two isolated fluid chambers.

As the cells cover and “seal” the small pores, they interrupt the direct path of electrical flow. Next, a perforating agent is introduced to create an electrical pathway through the portion of the cell’s membrane that is resting on the small pores. The associated electrode in the well allows you to clamp the membrane potential. At this point, the whole-cell ion-channel currents can be measured.

The IonWorks HT system integrates the use of the PatchPlate consumable with the operation of a sophisticated network of fluidics and electronics systems. The PatchPlate provides the crucial 384-well platform for partitioning and positioning the cells. Cells in suspension are added to individual wells of the PatchPlate by an integrated 12-channel pipettor. A fluidics network comprised of peristaltic pumps, solenoid valves and pressure regulators performs the PatchPlate priming, fluid exchange and wash operations. Electrical recordings of the ion channels are made with the 48-channel electronics head. The experiment stage houses the PatchPlate plenum along with soak and wash stations, cell and buffer reservoir positions and two compound plate stations.

1-2 IonWorks HT Users Guide

Overview of the IonWorks HT System

IonWorks HTSystem

Components

The main system components are listed below.

For a more complete description of the system components, refer to Chapter 2, “System Description.”

IonWorks HT System Components

♦ Cabinet that contains the whole system

♦ Experiment stage

♦ Integrated 12-channel fluidics head (F-Head)

♦ Integrated 48-channel electronics head (E-Head)

♦ Fluidics panel

♦ Personal computer running custom software that controls the instrument parameters, records data and transfers acquired data to spreadsheet-compatible files

Operating theIonWorks HT

System

Optimizing the PatchPlate Usage

Experimental protocols can be designed to specify the desired PatchPlate coverage, sequence of priming operations, fluid-handling modes, electrical test and measurement parameters and user-defined metrics. The utility of the PatchPlate consumable can be optimized by selecting either whole, one-half or one-quarter PatchPlate coverage. The IonWorks HT system can be programmed for one or no compound addition.

Designing Experimental Parameters

Additionally, there is the capability to configure the duration of the compound incubation and the types of compound plates. Editing the electrical signal parameters is achieved by a versatile and user-friendly Waveform Editor. A family of command voltages along with pre- and post-measurement parameters can be defined while actively previewing the waveform.

Designing Analysis Metrics

Lastly, a set of post-measurement analysis metrics can be defined as part of the experiment protocol. Metrics provide the means of distilling information in cell measurements into a single number for each metric defined. Through the Metrics Editor dialog box, up to 20 simple or combined metrics can be defined. These metrics apply user-specified mathematical methods to individual traces to arrive at a single value for each metric. Metrics are computed as real-time data collection proceeds during the experiment and are used to perform post-run data analysis.

Viewing the Data

The IonWorks HT system software displays the electrophysiological recordings in a 48-cell format during an experiment run. Typical experiments consist of multiple electrical scans per cell. Data updates occur in real time. The scaling or zoom parameters can be changed on individual traces during data acquisition.

Viewing the Analysis

Upon completion of the experiment, the primary analysis window displays a summary of the experiment data along with a color-coded, interactive view of either the



PatchPlate or the compound plate. The binary color-coding scheme provides key information for each well in the plate view. Wells with valid cell recordings are readily distinguishable from those with invalid information. Wells which exhibit characteristics of interest may be highlighted as “hits” for screening purposes. Individual wells from the plate view can be selected to access the detailed data on a cell-by-cell basis in separate windows. Data is displayed as either electrophysiological measurements over time (time profile) or single-number metrics per individual well (statistics format).

The primary analysis window provides analytical and statistical tools. Post-run analysis can be conducted by selecting combinations of scans, metrics and thresholds. Additionally, there is the option of auto-exporting the data for post-run analysis. In this case, the data is stored in an ASCII text file format for input into a spreadsheet or data-base program.


IonWorks HT System Specifications and Requirements

IonWorks HT System Specifications and Requirements

ElectricalRequirements

PhysicalSpecifications and

Requirements

VacuumRequirements

The IonWorks HT requires vacuum to operate. This can be provided by house vacuum or by purchasing a vacuum pump. House vacuum must meet the following specifications:

♦ 120 mBar @ 0.6 m/hr or 26 in. Hg @ 0.35 cm

♦ Have fittings required to attach to a 1/4'' plastic tubing

Alternatively, a Molecular Devices Corporation Vacuum Pump may be purchased.

Item/Parameter Description/Value

Line Voltage 120 VAC or 240 VAC instrument versions

Note System power supply is configured when manufactured.

Item/Parameter Description/Value

Dimensions The IonWorks system cabinet is 55.6 in. (H) x 58.1 in. (W) x 32.5 in. (D) (with access door closed).

Instrument setup space An additional 18 in. on each side is required to accommodate the source and waste container holding racks.

A minimum of 6 in of space must be maintained at all times in the back of the instrument.

A minimum of 32 in. is required to allow clearance for opening the access door.

Additional setup space An additional benchtop that is 36 in. wide, 30 in. long and 36 in. high is required for the host computer, display monitor and peripheral accessories.

Item Part Number

IonWorks HT vacuum pump, 120 VAC 3900-0025

IonWorks HT vacuum pump, 230 VAC 3900-0040



Safety

DocumentationUser Attention

Words

Five user attention words appear in the text of all Molecular Devices Corporation user documentation. Each word implies a particular level of observation or action as described below.

Note Calls attention to useful information.

IMPORTANT Indicates information that is necessary for proper instrument operation.

CAUTION Cautions the user that a potentially hazardous situation could occur, causing injury to the user or damage to the instrument, if this information is ignored.

Warns the user that serious physical injury to the user or other persons could result if these precautions are not taken.

Chemical HazardWarning

CHEMICAL HAZARD. Some of the chemicals used with Molecular Devices instruments are potentially hazardous and can cause injury or illness.

♦ Read and understand the material safety data sheets (MSDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials.

♦ Minimize contact with and inhalation of chemicals. Wear appropriate personal protective equipment when handling chemicals (e.g., safety glasses, gloves, or clothing). For additional safety guidelines consult the MSDS.

♦ Do not leave chemical containers open. Use only with adequate ventilation.

♦ Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer’s cleanup procedures as recommended on the MSDS.

♦ Comply with all local, state/provincial, or national laws and regulations related to chemical storage, handling, and disposal.

\

Chemical WasteHazard Warning

CHEMICAL WASTE HAZARD. Wastes produced by Molecular Devices instruments are potentially hazardous and can cause injury or illness.

♦ Read and understand the material safety data sheets (MSDSs) provided by the manufacturers of the chemicals in a waste container before you store, handle, or dispose of chemical waste.

♦ Handle chemical wastes in a fume hood.

♦ Minimize contact with and inhalation of chemical waste. Wear appropriate personal protective equipment when handling chemicals (e.g., safety glasses, gloves, or clothing).

About MSDSs Some of the chemicals used with this instrument may be listed as hazardous by their manufacturer. When hazards exist, warnings are prominently displayed on the labels of all chemicals.

Chemical manufacturers supply a current MSDS before or with shipments of hazardous chemicals to new customers and with the first shipment of a hazardous chemical after an MSDS update. MSDSs provide you with the safety information you need to store, handle, transport and dispose of the chemicals safely.

We strongly recommend that you replace the MSDSs in your files each time you receive one packaged with hazardous chemicals.

WARNING!

WARNING!

WARNING!


Safety

CHEMICAL HAZARD. Be sure to familiarize yourself with the MSDSs before using reagents or solvents.

Ordering MSDSs To order an MSDS for a chemical, call the chemical manufacturer.

Instrument SafetyLabels

Safety labels are located on the instrument. Each Safety label consists of a:

♦ Signal Word panel, which implies a particular level of observation or action (e.g., CAUTION or WARNING). If a safety label encompasses multiple hazards, the Signal Word corresponding to the greatest hazard is used.

♦ Message panel, which explains the hazard and any user action required.

♦ Safety Alert symbol, which indicates a potential personal safety hazard.

Before Operatingthe Instrument

Ensure that everyone involved with the operation of the instrument has:

♦ Received instruction in general safety practices for laboratories

♦ Received instruction in specific safety practices for the instrument

♦ Read and understood all related MSDSs

CAUTION Avoid using this instrument in a manner not specified by Molecular Devices Corporation. While the instrument has been designed to protect the user, this protection may be impaired if the instrument is used improperly.

CAUTION Use of controls or adjustments or performance of procedures other than those specified herein may result in hazardous UV exposure.



Safety Alert Symbols

Electrical Symbols The following chart is an illustrated glossary of all electrical symbols that are used on Molecular Devices instruments. Whenever such symbols appear on instruments, please observe appropriate safety procedures.

NonelectricalSymbols

The following is an illustrated glossary of all nonelectrical safety alert symbols found on Molecular Devices instruments.

This symbol indicates the ON position of the main power switch.

This symbol indicates the OFF position of the main power switch.

This symbol indicates the ON/OFF position of a push-push main power switch.

This symbol indicates that a terminal may be connected to another instrument’s signal ground reference. This is not a protected ground terminal.

This symbol indicates that this is a protective grounding terminal that must be connected to earth ground before any other electrical connections are made to the instrument.

A terminal marked with this symbol either receives or delivers alternating current or voltage.

A terminal marked with this symbol can receive or supply an alternating and a direct current or voltage.

CAUTION This symbol indicates the presence of high voltage and warns the user to proceed with caution.

CAUTION This symbol alerts you to consult the manual for further information and to proceed with caution.

~~

CAUTION This symbol illustrates a heater hazard. Proceed with caution when working around these areas to avoid being burned by hot components.

This symbol indicates that a laser is present inside the instrument.


2
System Description 2
Overview

In This Chapter This chapter provides detailed information about the IonWorks™ HT system components.

Topic See Page

IonWorks HT System Components 2-2

System Cabinet 2-3

Experiment Stage 2-4

12-Channel Fluidics Head 2-7

48-Channel Electronics Head 2-8

Electronics Cabinet 2-10

Fluidics Panel 2-11

Source and Waste Bottle System 2-13

System Computer 2-15


Chapter 2 System Description

IonWorks HT System Components

Overview The IonWorks HT system is comprised of the following components:

♦ System cabinet


♦ Integrated 12-channel fluidics head

♦ Integrated 48-channel electronics head

♦ PatchPlate consumable

♦ Electronics

♦ Fluidics panel

♦ Source and waste containers

♦ Personal computer running custom software that controls the instrument parameters, records data and transfers acquired data to spreadsheet-compatible files

IonWorks HTSystem

Below is a picture of the Molecular Devices IonWorks HT System with the components labeled.

Source bottles and holding rack

Experiment stage, 12-channel fluidics head and 48-channel electronics head

Waste bottles and holding rack

Power switch and indicator

Hinged access door

System cabinet

Electronics compartments

Fluidics panel


System Cabinet

System Cabinet

Cabinet UpperCompartment

The system cabinet has two main compartments. The upper compartment is accessed by lifting the hinged access door.

The upper compartment houses:


♦ Integrated 12-channel fluidics head

♦ 48-channel electronics head

♦ Upper debubbler

Cabinet LowerCompartment

The lower compartment houses:

♦ Fluidics panel

♦ Electronics

Note There are installed safety interlocks that terminate any system operation once the front access door is opened.

Source and WasteContainers

The left- and right-side exteriors of the system cabinet provide holding racks for sets of input and output carboy bottles. The cabinet is supplied with casters and leveling feet for facilitating the instrument transport and for securing the system in a stationary position.



Experiment Stage

Components ofthe Experiment

Stage

The experiment stage has the following sub-components:

♦ Electronics head soak station

♦ Electronics head wash station

♦ PatchPlate station with clamping frame

♦ Fluidics head wash station

♦ Compound plate 1

♦ Cells suspension reservoir

♦ Buffer reservoir

♦ Compound plate 2

Removable Reservoirs

There are two types of removable reservoirs used with the experiment stage:

♦ V-bottom

♦ Flat-bottom

The V-bottom reservoir has a capacity of 5 mL and can be used interchangeably in either the CELLS or the BUFFER positions. The flat-bottom reservoir has a capacity of 10 mL and is used exclusively in the SOAK position.

IMPORTANT Placing the V-shaped reservoir in the SOAK position will cause damage to the E-Head.

Electronics head soak station

Electronics head wash station

PatchPlate and clamping frame

Cells station

Fluidics head wash station

Compound plate 1

Buffer station

Compound plate 2

Experiment stage


Experiment Stage

Prior the start of an experiment, these reservoirs are positioned in appropriate stations on the experiment stage and are loaded with the proper solutions. After the experiment is completed, the solutions are discarded and the reservoirs are cleaned with ethanol solution.

Electronics-HeadSoak-Station

The electronics-head soak-station houses the removable flat-bottomed reservoir that is used for soaking all 48 probes of the electronics head simultaneously. This station is used for functions such as applying a chloride coating to all electrodes (see “Chloriding the E-Head Probes” on page A-4). The electronics head soak station is labeled SOAK on the experiment stage.

Electronics-HeadWash-Station

This station is an embedded reservoir that contains 48-wash channels for isolating and rinsing the probes of the electronics head. All 48 channels of the embedded reservoir circulate a fresh supply of wash solution for rinsing the electronics head probes. A peristaltic pump on the fluidics panel serves as the main agent for directing the flow of the wash solution from the source bottle to the wash station. The embedded wash reservoir contains an exit drain which is connected to the waste bottle. A solenoid pinch-valve opens the drain to the system vacuum line which in turn empties the contents of the embedded reservoir. The source bottle for the electronics-head wash-station is labeled E-Head Wash.The waste bottle for the electronics-head wash-station is labeled Wash Waste. The source and waste bottles are located at the left- and right-side of the system cabinet, respectively.

PatchPlate Stationwith Clamping

Frame

To perform experiments, the PatchPlate must be loaded and sealed into the IonWorks HT system. This station is where the PatchPlate consumable is loaded. This station is the central location for experimental operations which include fluid pipetting, plate priming, fluid exchange and electrical recordings. A clamping frame is used to vacuum-seal the PatchPlate consumable to the station.

The mechanical block that houses the PatchPlate is called the plenum. The plenum contains a cavity for loading the PatchPlate. This cavity houses a ground bath electrode and a small port for monitoring the plenum vacuum pressure as well as plenum fluidics into and out of the ports. The top surface of the plenum contains an embedded o-ring and a vacuum port. The o-ring outlines the perimeter of the PatchPlate cavity and is used for sealing the PatchPlate to the plenum. The vacuum port is connected to a switch for releasing and holding the PatchPlate clamping frame. The clamping frame rests on the borders of the PatchPlate. Once activated, the vacuum port provides the means for sealing the clamping frame to the plenum. The vacuum-sealed clamping frame in turn exerts evenly-distributed force to seal the PatchPlate to the plenum. The PatchPlate vacuum switch is located on the main platform of the IonWorks HT system and can be turned to two positions. These positions are labeled RELEASE and HOLD.

Source and WasteBottles for the

PatchPlate Station

There are two source bottles for the PatchPlate station. The main source bottle is labeled Internal Buffer. The source bottle for fluids exchange operations such as introduction of a perforating agent is labeled Antibiotic. The waste bottle is labeled Plenum Waste. The source and waste bottles are located on the left- and right-side of the system cabinet, respectively.



Fluidics-HeadWash-Station

This station is an embedded reservoir with 12-wash channels for isolating and cleaning the pipettor needles. All 12 channels of the embedded reservoir circulate a fresh supply of wash solution for rinsing the fluidics-head needles. A peristaltic pump on the fluidics panel serves as the main agent for directing the flow of wash solution from the source bottle to the wash station. The embedded wash reservoir contains an exit drain which is connected to the waste bottle. A solenoid pinch-valve opens the drain to the system vacuum line which in turn empties the contents of the reservoir.

Source and WasteBottles for theF-Head Wash

The source bottle for the fluidics head wash station is labeled F-Head Wash. The waste bottle for fluidics head wash station is labeled Wash Waste. The source and waste bottles are located at the lett- and right-side of the system cabinet, respectively.

Compound Plates The experiment stage accommodates up to two compound plates. The compound-plate positions are labeled on the experiment stage as PLATE 1 and PLATE 2. Either 96- or 384-well microplates can be loaded in these positions.

Note The plate manufacturer and plate type must be specified and selected from a list provided through the IonWorks HT system software prior to instrument operation.

Cells SuspensionReservoir

The cell-suspension reservoir area houses a removable v-bottom reservoir, or a 12-well cell boat (for cell-line optimization), which are used for holding the cell suspension. During an experiment, the 12-channel fluidics head aspirates the cell suspension from this station and dispenses it into the PatchPlate. This station is labeled CELLS on the experiment stage.

Buffer Reservoir The buffer-reservoir area houses a removable v-bottom reservoir which is used for holding a fresh supply of appropriate buffer solution. During an experiment, the 12-channel fluidics head aspirates the buffer solution from this station and dispenses it into the PatchPlate. The buffer station also serves as the main station for rinsing the 12-channel fluidics head during “flush and rinse” operations. This station is labeled BUFFER on the experiment stage.


12-Channel Fluidics Head

12-Channel Fluidics Head

Features of theFluidics Head

The 12-channel fluidics head is designed for pipetting into the PatchPlate. The spacing of the fluidics head needles is compatible with both 96- and 384- well microplates.

The fluidics head can be used to load either all or a portion (1/4 or 1/2 of the PatchPlate) of the 384-wells in a PatchPlate consumable. Depending on the chosen PatchPlate-consumable coverage, operations such as loading cells may require pipetting into all 384 wells. The fluidics head has the capacity for loading all 384 wells of the PatchPlate consumable in a single-aspirate and multiple-dispense cycle.

The fluidics head is equipped with motion axes that provide mobility and expedite the pipetting operations. The motion axes allow the fluidics head to access either the PatchPlate consumable or any of the fluidics stations and compound plate positions on the experiment stage.

The fluidics head can also perform pipetting operations and mixing functions following compound additions to the PatchPlate.

Fluidics HeadMechanism of

Action

The design of the fluidics head incorporates 12 pistons that move in their individually isolated cylinders. Each cylinder is sealed at one end and open at the other. The underlying mechanism for the operation of the fluidics head is air displacement. As pistons move within their cylindrical housing, they displace a volume of air. The air displacement either creates a vacuum for aspirating fluid into the individual tips or exerts a force that dispenses the fluid out the individual tips. The motion of the pistons is controlled by stepper motors and encoders to maintain the required pipetting performance.

Note The fluidics head performance is designed to meet the precision and accuracy required of industry-standard micro-pipetting systems.



48-Channel Electronics Head

Features of theElectronics Head

CAUTION DO NOT CONTACT the electronics head and its individual probes. While the electronics head housing provides a shielding mechanism against electro-static discharge (ESD) to the enclosed circuit boards, the individual probes are extremely susceptible to damages caused by ESD.

The 48-channel electronics head is designed for recording electrical measurements from the 384 wells of the PatchPlate plenum. The electronics head is comprised of 48 silver-plated probes that access individual wells of the PatchPlate to measure electrical currents simultaneously.

Each probe is connected to a separate amplifier on an enclosed printed circuit-board inside the electronics-head housing. Each electronics head is calibrated across all 48- probes to normalize performance. A chloride coating is applied to all 48 probes periodically by soaking them using the electronics soak station.

The electronics head is equipped with motion axes that allow the head to access either the PatchPlate or the electronics wash and soak stations.

Note The electronics head performance matches the high-fidelity measurement levels demanded of industry-standard patch-clamp systems.

Electronics HeadMechanism of

Action—TheAmplifier System

There are 48 individual high-gain transimpedance amplifiers arranged in 4 banks of 12 and are enclosed in the electronics-head housing. These amplifiers perform the same basic current-to-voltage conversion as the head-stage in a classical patch-clamp apparatus.

All operations on the IonWorks HT are performed in a voltage-clamp mode. Command and hold voltages which are specified in the software and generated by a digital-to-analog converter in the control module, are routed to the head. Output signals from the amplifiers are multiplexed onto a cable in groups of 12 and returned to the control module where they are digitized. All probes are maintained at the user-specified hold voltage when not being actively read. During a read, the user-specified test waveform is applied to one bank at a time until all four banks have been read.

Note There are no user-serviceable parts on the amplifier boards.

CAUTION Do not remove the cover as the boards are susceptible to damage from static discharge.


PatchPlate Consumable

PatchPlate Consumable

Overview The PatchPlate consumable provides a 384-well platform for obtaining simultaneous, parallel electrical recordings. The PatchPlate is composed of two main components:

♦ 384-well plate—manufactured as an injection-molded piece

♦ Bottom piece—piece that contains 384 small pores

Well Arrangementand Capacity

The 384 wells of the PatchPlate are arranged in a grid of 8-rows by 48-columns. The capacity of each individual well is 12µL.

The wells of the plate are recognized by the instrument as follows:

♦ The 8-rows of the PatchPlate are distinguished by letters i.e., A–H

♦ The 48-columns of the PatchPlate are distinguished by integers i.e., 1–48.

Individual wells, therefore, are distinguished by an arrayed designation. When the PatchPlate is loaded into the PatchPlate station on the IonWorks HT experiment stage, the well closest to the front-left side of the overall system cabinet is A1. Consequently the well closest to the rear-right side of the system cabinet is H–48, the 384th well. Refer to the diagram of the experiment stage shown below.

PatchPlatePackaging

The PatchPlate consumables are packaged in groups of five. The clear plastic packaging is heat-sealed to an opaque plastic lid with perforations that allow removal of one PatchPlate at a time. Each PatchPlate box contains 50 PatchPlates (10 x 5 packs). The PatchPlates must be stored in a cool, dry place. The PatchPlate performance is assured for six-months from the date of receipt.

Compound Plate 1 Compound Plate 2

Buffer S

tation

Cell S

tation

Fluidics H

ead Wash S

tation

E-H

ead Wash S

tation

E-H

ead Soak S

tation

A B C D E F G H

1

384

PatchPlate



Electronics Cabinet

Components ofthe Electronics

Cabinet

Located at the lower-right compartment of the system cabinet, the electronics cabinet houses three modules:

♦ Power supply

♦ Control

♦ Motor drive

Power SupplyModule

The cabinet drawer with the main-system power button is the power-supply module. It contains all the electrical power required for the IonWorks HT system.

Note The power-supply module is the only component that must be configured to the end-user’s voltage settings (110 VAC /4A or 220 VAC /2A) prior to the systems shipment.

Control Module This serves as the central control location for coordinating system operations. The control module houses the IonWorks HT embedded computer system along with digital and analog cards for various input/output functions. It accepts the external ethernet line as well as the internal connections for the electronics head, fluidic valves, vacuum regulator and motion controllers.

Motor DriveModule

This module coordinates the electronics- and the fluidics-head motion operations as well as the fluidics pump-actions. The motor-drive module houses a central motor interface-board that interacts with the enclosed stepper-motor and peristaltic-pump drives. It operates the system safety-interlock for the access window along with connections for the various motor-, pump-drives and encoders.


Fluidics Panel

Fluidics Panel

Peristaltic Pumps Located at the lower-left compartment of the system cabinet, the fluidics panel performs priming, fluid exchange and wash operations. The fluidics panel is comprised of a network of interconnected tubing with inline peristaltic pumps, solenoid valves and vacuum regulators. There are three integrated peristaltic-pumps:

♦ Fluidics wash

♦ Electronics wash

♦ Plenum

Fluid Control andNetworking

As the pumps circulate the solutions in separate tubing pathways, a series of inline solenoid pinch-valves control the direction of fluid flow. These valves also perform a pulsing sequence during the priming operations to clear bubbles from the system. Housed at either side of the system cabinet, input and output bottles provide the source and the waste containers for the fluidics panel. One of the output bottles provides the entry point for the system’s main vacuum line. The vacuum line is routed to the following locations:

♦ PatchPlate plenum

♦ Vacuum regulator

♦ Fluidics-head wash-station

♦ Electronics-head wash-station

Plenum andDebubblers

The plenum provides a vacuum-sealed interface for loading the PatchPlate and for performing plate operations such as priming, debubbling and exchanging fluids.

A clamping frame, along with a vacuum-entry orifice on the plenum’s top surface, is used to seal the patch-plate. A pressure gauge on the fluidics panel monitors the patch-plate plenum seal and reports the vacuum pressure (in mm Hg).

The vacuum-sealed lower chamber that is created at the underside of the PatchPlate allows for various priming and fluid exchange operations. The plenum contains input and output ports that are connected to source and waste bottles respectively. A tubing network with inline peristaltic-pump and pinch-valves feed the input port of the plenum. Appropriate solution can be circulated through tubing lines to prime and to remove any air bubbles. Two integrated vessels provide inline traps for removing and collecting air bubbles from the plenum tubing lines. Referred to as debubblers, these vessels are connected to the computer-controlled vacuum regulator that is integrated with the fluidics panel. Consequently the debubblers serve as control points for regulating the plenum differential pressure. The upper debubbler is located at the left-side of the experiment stage and can be used as a quick user-feedback for monitoring the plenum fluidics operations. After completion of priming, debubbling and/or fluids exchange operations, the fluid height at the upper debubbler must reach the appropriate level. Fluid heights that fall outside the appropriate range may indicate a problem with the fluidics panel.



Vacuum Regulator The integrated vacuum regulator adjusts the vacuum pressure to required levels for priming the PatchPlate and adding cells. A digital vacuum gauge on the fluidics panel displays the actual differential vacuum pressure (inches of water). The vacuum is also used to empty the contents of both the fluidics head and the electronics head wash stations.


Source and Waste Bottle System

Source and Waste Bottle System

Components The source and waste bottles are located at the left- and right-sides of the system cabinet, respectively. The source bottles supply the system with fresh solutions while the waste bottles collect the fluid waste.

Source Bottles Four source bottles are housed on a positioning rack that is installed on the left-side of the system cabinet. The table below describes the contents and properties of these source bottles:

Each bottle is mated to a threaded cap that is distinguished by appropriate labeling. The caps contain filling ports that contain barbed fittings and are connected to tubing lines. The tubing lines access the contents of the source bottle at one end. They provide a continuous path to the appropriate station on the experiment stage and terminate at the waste bottles at the other end.

Note Source bottles must be housed at the correct positions on the holding rack.

Waste Bottles Two waste bottles are housed on the holding rack at the right-side of the system cabinet. The vacuum waste bottle has a capacity of 4 L and collects the fluid waste from the various wash stations on the experiment stage. The plenum waste bottle has a capacity of 4 L and collects the fluid waste from the patch-plate station.

Each bottle is mated to a threaded cap which contains filling ports. The filling ports are connected to tubing lines that transport the waste fluids from the various stations to the appropriate source bottle. One of the filling ports on the vacuum waste bottle cap

Source BottleCapacity

(L)Dead Volume

(mL) Contents

F-Head Wash 2 100 F-Head wash solution

E-Head Wash 1 50 E-Head wash solution

Internal Buffer 2 100 Internal buffer solution

Antibiotic 1 50 Perforation agent in internal solution

F-Head wash

E-Head wash

Internal buffer

Antibiotic



serves as the entry point for the systems main vacuum line. The two-position holding rack contains several installation slots to allow for adjusting the height of the waste bottles. The waste bottles are part of the closed vacuum system and provide a reference point for setting the plenum differential pressure. The height of the holding rack is set by the instrument manufacturer.

Wash-waste bottle

E-Head/F-Head waste bottle

Two-position holding rack

Main vacuum input line


System Computer

System Computer

ComputerConfiguration

The IonWorks HT system software runs on a computer with the following minimum configuration:

♦ Intel Pentium® 4 (1.8 GHz or above)

♦ 256 MB of RAM

♦ 40 GB hard disk drive

♦ 2 ethernet network interface cards

♦ 21” monitor at 1280 x 1024 resolution

♦ Windows 2000 or Windows XP Operating System


3
Starting Up and Shutting Down the System 3
Overview

In This Chapter This chapter provides the information and procedures required to properly start-up and shut-down the IonWorks® HT System.

Topic See Page

Starting-Up the IonWorks HT System 3-2

Shutting-Down the IonWorks HT System 3-3


Chapter 3 Starting Up and Shutting Down the System

Starting-Up the IonWorks HT System

About Starting-Upthe System

Prior to start-up of the IonWorks HT system, the instrument installation must be completed by a Molecular Devices Service Representative. In order to ensure proper communication between the IonWorks HT system control software and hardware, the start-up and shut-down procedures described in this chapter should be followed precisely.

Procedure forStart-Up

To start-up the IonWorks HT system:

Step Action1 Check and verify the instrument power cord is connected to the room outlet with the

specified requirements:

Note The IonWorks HT instrument connection for the power cord is located at the back of the power-supply module inside the electronics cabinet,

CAUTION The instrument main power switch (located on the front of the power-supply module in the electronics cabinet) must be in the OFF position prior to plugging in the power cord.

2 Check and verify the ethernet cable is connected between the IonWorks HT system and the computer.

Note The ethernet connection for the IonWorks HT is located at the back of the control module inside the electronics cabinet. The ethernet connection for the computer is on the ethernet card receptacle in the back of the computer unit and is labeled Instrument,

CAUTION The computer contains another ethernet connection for networking purposes. This ethernet connection is labeled Network. Do not plug the instrument ethernet cable to the network connection.

3 Check and verify the main line for the vacuum line is connected to the proper port on the wash waste bottle.

4 Check and verify the PatchPlate vacuum switch is set to the Release position.

5 Check and verify the instrument access door is closed and the handle turned to the lock position.

6 Turn on the vacuum pump, or if the system is connected to house vacuum, ensure that it is on and operating properly.

7 Turn on the system computer and monitor.

Login to the system and launch the IonWorks HT software by double-clicking the desktop icon.

8 Turn on the instrument by pressing the power button located on the front of the power-supply module in the electronics cabinet.

9 Wait until the ready traffic-light button is lit (green) in the main window toolbar before proceeding. This will take ~3.5 min.

10 If performing an experiment, proceed to Chapter 5, “Operating the System.”


Shutting-Down the IonWorks HT System

Shutting-Down the IonWorks HT System

Procedure forShutting-Down

Note This procedure should be followed after an experimental run and the system has been properly cleaned as described in Chapter 5, “Operating the System.”

To shut-down the IonWorks HT system:

Step Action1 At the end of the programmed experiment, wait for all operations to terminate, The

ready traffic-light on the main window toolbar should be lit green.

2 Check to make sure that a Flush and Rinse operation was performed with 50/50 ethanol/filtered water wash solution (see Chapter 5, “Operating the System.”)

3 Exit the IonWorks HT software by selecting Exit from the File menu.

4 If not using house-supplied vacuum, turn off the vacuum pump.

5 Check and verify the following:

♦ The PatchPlate vacuum switch on the experiment stage is set to Release

♦ All removable reservoirs have been removed and cleaned

♦ A used PatchPlate is positioned in the plenum to exclude dust or particulates

♦ The instrument access door is closed and the handle is turned to the lock position


4
System Software Description 4
Overview

In This Chapter This chapter provides a detailed description of the features and functions of the IonWorks™ HT System software.

Topic See Page

Main Window 4-2

Main Window with View PatchPlate Selected 4-4

IonWorks Software Menu 4-5

Main Window with View Compound Plate Selected 4-16

Dialog Boxes and Windows 4-19

File Menu Dialog Boxes 4-20

Experimental Protocol Properties Dialog Boxes 4-24

View Menu Dialog Boxes and Windows 4-46


Chapter 4 System Software Description

Main Window

IonWorks SoftwareMain Window

The main window of the IonWorks software includes a Title bar, Menu bar, Toolbar, and Status bar.

There are two views of the main window, one view is displayed when View PatchPlate is selected and another view is displayed when View Compound Plate is selected. Examples of both are shown below.

View PatchPlate

Title bar

Menu barToolbar

Status barPatchPlate

Configure filter metrics

Configure analysis method

Plate legend and statistics

Configure hits


Main Window

View Compound Plate

Compound plateResults from compound plate



Main Window with View PatchPlate Selected

Title Bar

The title bar extends across the top of the window, inside the window borders. It displays the name of the application followed by the name of the assay or data file when one is open. The color of the title bar indicates whether or not it is the active window: the title bar of an active window is typically a different color (and usually brighter) than other window title bars for programs that are inactive (which may be dimmed).

Dragging the title bar repositions the window on the screen (in window view only; if the window has been maximized, dragging will not work). The buttons that appear at the right end of the title bar can be used to minimize the window so that it appears only on the task bar, maximize the window to full screen, or to close the window.

Status Bar This area displays the tool tip for an object when the cursor is placed over it and otherwise displays the status of the instrument.

The panel to the right displays the name of the active protocol.


IonWorks Software Menu


Menu Bar

The menu bar is the area across the top of the window directly below the title bar. It contains the names of the menus that group together related commands. The File and Edit menus control the export, save, and print functions, and the ability to modify protocols and experimental metrics.

The View menu allows you to view the protocol and experimental results.

The Experiment menu controls the start and stop of experiments and allows you to monitor the experiment in real-time.

The Utilities menu enables you to control the instrument hardware.

The Window and Data menus (only available when a data file is open) allow you different viewing options for the data.

The Help menu displays information about the version of IonWorks software and provides access to the system user manual.

Clicking on a menu name displays a list of commands that can be used to access software functions. The contents of these menus are described below.

File Menu

A description of the menu items are listed below:

Item Description

Open Data Opens a previously saved data (*.ihe) file.

Close Closes the current data file.

Save Saves the currently active data file.

Save As Saves the data file with a new name.

Open Protocol Opens a protocol from either a previously saved protocol (*.ihp) or data file.

Save Active Protocol Saves the currently active protocol



Edit Menu

\

A description of the Edit menu items is listed below:

View Menu

A description of the View menu items is listed below:

Export Traces Opens the Configure Trace Export dialog box which allows you to export the data as an ASCII text file for multiple wells of an experiment. The results are exported a an ASCII text file (*.ihd).

Export Metrics Opens the Export Metric dialog box and allows you to export the analysis results from the metrics that are defined in the experiment. The results are exported as an ASCII text file (*.iha).

1, 2, 3... The data files (up to 10) that were opened most recently. The most recent file is labeled 1.

Exit Closes the program application. If you have unsaved data, you will be prompted to save it before closing.

Item Description

Item Description

Protocol Opens the Experimental Protocol Properties dialog box to allow editing of the experimental parameters.

Metrics Opens the Edit Metric Menu dialog box to allow you to create, edit or delete analysis measurements of the experimental protocol.

Note This command only appears when a data file is open.

Filters Opens the Filter Editor dialog box to allow you to change the metrics of the filters.

Item Description

Toolbar When checked, displays the main window toolbar.

Status Bar When checked, displays the status bar.

Active Protocol Opens the Summary of Active Protocol dialog box.

Experiment Summary Opens the Experiment Summary dialog box.

Trace Groups Opens a display view of the signal traces of the data file.



Experiment Menu

A description of the Experiment menu items is listed below:

Note The Experiment menu items are disabled unless the IonWorks HT instrument is connected and powered on.

Utilities Menu

A description of the Utilities menu commands is listed below:

Note The Experiment menu items are disabled unless the IonWorks HT instrument is connected and powered on.

Messages and Logging Opens the IonWorks Message dialog box which displays status messages from the instrument during operation. Message logging to an ASCII text file can be set up and activated from this dialog also.

Item Description

Item Description

Run Starts the experimental protocol.

Stop Stops the experimental protocol.

Monitor Opens the Monitor dialog box that displays status messages from the instrument and indicates the time remaining in the experiment. The Trace Groups window can be opened from the Monitor dialog box to display the data in real time.

Command Description

Flush and Rinse Flushes the tubing in the vacuum system and rinses using buffer.

Wash Electrodes Moves the electrodes to the wash station and executes the wash process.

Soak Electrodes Moves the electrodes to the soak (chloriding) station and soaks the electrodes.

Park Electrodes Moves the electrodes back to the home position.

Test Electrodes Opens the electrode testing dialog box.

Wash Pipettor Moves the pipettor to the wash station and executes the wash process.



Window Menu Note This menu is only available when a data file is open.

A description of the Window menu Commands is listed below:

Data Menu Note This menu is only available when a data file is open.

A description of the Data menu items is listed below:

Park Pipettor Moves the pipettor back to the home position.

Test Pipettor Opens the Pipettor Validation dialog box.

Stop Stops the utility operation in progress.

Command Description

Command Description

New Window Opens another window on the currently selected data file.

Cascade Arranges the currently open windows in a cascade pattern.

Tile Arranges the currently open windows by tiling the main window.

Arrange Icons Arranges the minimized windows.

1–10 (data files) Selects an open data-file window and brings it to the foreground.

Item Description

Leak Correction Enables/disables leak correction.

Offset Current Opens a dialog box that allows the offset current feature to be enabled and the region for the determination of the offset to be specified. The offset current feature is used to correct for minor differences in the non-stimulated ion current of the cells. This feature is disabled by default.



Help Menu

A description of the Help menu items is listed below:

Toolbar

Each button on the toolbar represents a menu command and is a shortcut to activating that command.

Data File Title Bar The data file title bar extends across the top of the opened data file inside the main window. The data file title bar shows the following information:

♦ Name of the file

Item Description

About IonWorks HT Opens a dialog box that displays the version of the IonWorks HT system operating software and the version of the firmware currently running on the instrument.

User Manual Opens a PDF version of the current IonWorks HT System User Manual.

The buttons on the toolbar are as follows:

Button Name Description

Open file Opens a data file.

Save file Saves the data file currently showing on-screen.

Start experiment Starts the experimental protocol.

Stop experiment Stops the experimental protocol.

Messages Opens the IonWorks message dialog box.



PatchPlate Area Displays a schematic of the 384-well PatchPlate. The red-colored wells have been excluded from the determination of “Hits” i.e., wells of interest based on the filtering criteria shown in the plate legend and statistics area. The yellow-colored wells indicate “Hits” and are defined in the Configure Hits Display pane.

Note Double-clicking on a well in the PatchPlate area will open a window that displays the current readings graphically. See “Viewing Compound Plate Well Data” on page 4-17 for more information about this feature.



Plate Legend andStatistics Area

This area defines the excluded wells indicated by color (red), +/- X on the PatchPlate and displays statistical information regarding the remaining, non-excluded wells.

The items in this area are defined as follows:

Item Description

Show Valid-Cell Count

Active in Compound Plate view only. Each compound is run in quadruplicate on the PatchPlate. When active, it will identify the total number of successful experimental runs from the PatchPlate.

Total Displays the total number of cells (or wells) in the viewed plate format.

Indicates the number of wells that failed to pass the seal resistance metric for the currently selected scan. If the selected scan is a Seal Test scan, then the failure occurred during the seal test measurement. If the selected scan is a user-defined scan such as the Pre-Compound scan, then the seal resistance is determined during the pre-scan period of the command voltage protocol.

Displays the number of cells (or wells) that were excluded on a basis of options chosen in the Configure Metrics Filter pane.

Mean Average value of the remaining, non-excluded cells (or wells). The value of this number will depend on the chosen analysis method.

Median Comparing only non-excluded cells, the value for which there are equal number of values above and below it.

Std Dev The standard deviation of the values for all non-excluded cells.

Min The well location and value of a cell with the lowest non-excluded value.

Max The well location and value of the cell with the highest non-excluded value.



Configure FilterMetrics Pane

This pane of the main window displays the mathematical measurement choices defined by the user. They will be applied to the data for the purpose of setting a threshold exclusion thus defining the exclusion limits for a cell. Click on Edit Filters to change the measurement choices for a particular filter and allow recalculation of the data.

.

Below is a description of the items and choices in this pane.

Item Description

Enable 1.2.3.4.5 When checked, the filter metric is enabled and can be defined. To define a filter, click Edit Filter and select the desired options (see “Edit Filter Dialog Box” on page 4-43).

User-defined filters Indicates which filter metrics are enable and defined. Filter metrics can be modified by opening the Edit Filters dialog box.

Disable All User-Defined Filters

When checked, no filter metrics will be applied to the data, regardless of what is selected in this pane.

When clicked, the Edit Filters dialog box opens. See “Edit Filter Dialog Box” on page 4-43 for more information about this dialog box.



ConfigureAnalysis Method

Pane

This pane of the main window displays the choices for the analysis method that will be used to analyze the PatchPlate read.

Below is a description of the items and choices in this pane.

Item Description

Display Statistics Based on a Single Scan

When selected, the statistics will be based on the scan chosen from the adjacent drop-down menu. The scans to choose from will always include the Hole Test and Seal Test, but depending on the type of experiment, there may be more or less than two additional scans. For the single compound addition, there will be two scans labeled Pre-compound and Post-compound, but if the Interactive Scan mode is used, there can be one or more scans labeled Scan #1 etc.

Metric Displays the analysis method used for analyzing the data of a single scan. The resistance and offset voltage metrics are present for all experiments and do not appear in the Edit Metric dialog box even though they appear in this list. Another example of a metric that may appear in this list and in the Edit Metrics dialog is the kv Metric.

Combine metrics within a well using the

Average, minimum or maximum

Display Statistics Based on Two Scans

When selected, the statistics will be based on two scans chosen from the adjacent drop-down menus of Scan A and Scan B. The scans to choose from will always include the Hole Test and Seal Test, but depending on the type of experiment, there may be more or less than two additional scans. For the single compound addition, there will be two scans labeled Pre-compound and Post-compound, but if the Interactive Scan mode is used, there can be one or more scans labeled Scan #1 etc.



Configure HitsDisplay

This pane contains the options that can be used for displaying cell wells that are “Hits”.

Below is a description of the items and choices of the Configure Hits Display pane.

Metric Displays the analysis method used for analyzing the data of two scans. The resistance and offset voltage metrics are present for all experiments and do not appear in the Edit Metric dialog box even though they appear in this list. Another example of a metric that may appear in this list and in the Edit Metrics dialog is the kv Metric.

Cross scan comparison method

Displays the analysis method that will be used for comparing the two scans. The choices are:

♦ % Difference: 100*(Scan B - Scan A)/Scan A

♦ Absolute Value of % Difference

♦ Difference: Scan B - Scan A

♦ Absolute Value of Difference

♦ Ratio: Scan B/Scan A

♦ Absolute Value of Ratio

Combine metrics within a well using the

Average, minimum or maximum.

Item Description

Item Description

Display hits for metrics that are less or equal to /greater than this value

Cell wells that meet the criterion entered in these fields will be indicated by the color yellow in either the PatchPlate or the Compound Plate diagrams.

When clicked, the data is recalculated using the displayed settings in this window.

Displays the number of hits detected using the criterion set in this window.



Display hits for two scan comparisons less/greater than tis value

Cell wells that meet the criterion entered in these fields will be indicated by the color yellow in the PatchPlate area.

Note This option is available when two scan statistics is enabled in the Configure Analysis Method pane.

Note When determining a threshold value for use with the Hits Display, be aware that the values displayed in the Plate Legend and Statistics Area are rounded to three decimal places, whereas the values used by the Hits Display are much higher precision. Values greater than three decimal places can be specified for the hits threshold.

Item Description



Main Window with View Compound Plate Selected

Compound PlateArea

This view is displayed when View Compound Plate is selected.

Note This option is only available when the Single Addition mode was used for the experiment.

In the example shown, the compound plate type was a 96-well microplate.

Wells that are filled with yellow color indicate that they have passed all of the filter criteria in the Filter Metric and the Configure Analysis panes and passed the threshold in the Configure Hits pane. When the pointer is placed over the well (as in C3) the data results for that compound well are displayed to the left of the compound plate in the main window (see below). In this case, since the compound plate has 96-wells, each well will deliver compound to four different cell wells of the 384-well PatchPlate.

When Show Valid Cell Count is selected, the number in the well indicates the number of PatchPlate wells that have passed all enabled User-defined Metric filters as well as the Native Filter for cell seal resistance.

Microplate row

Microplate column

Well C3 with pointer to display data results

Data results for compound well C3


Main Window with View Compound Plate Selected

ViewingCompound Plate

Well Data

Double-clicking on a well in the compound plate area will open a window that displays the current readings pre- and post-compound graphically.

Compound Plate Window Menu Commands

File Menu Command Description

Exit Close the window and returns to the main window

Plot Menu Commands Description

Scaling Opening the Scaling dialog box and allows you to change the axes of the graphs.

Apply scaling globally The scaling options specified in the Scaling dialog box for this window will be applied to all other Compound Plate windows. This feature can be used to set all of the windows to autoscale or to force the plot scales to the same set of manually-entered values.

Indicates compound plate well

Indicates patchplate wellGraph legend

Command voltage



Viewing theAnalysis Metrics

Summary

The compound-plate well data also displays the analysis metrics for the indicated PatchPlate wells in tabular form.

Show/Hide Command Voltage

Toggles between displaying and hiding the command voltage applied during the experiment.

File Menu Command Description

Metric summary data

Analysis metric


Dialog Boxes and Windows

Dialog Boxes and Windows

Becoming Familiarwith Dialog Boxes

and Windows

If you've worked with other applications that run using Windows, you'll probably be familiar with the conventions in this section. For a more detailed discussion of windows and dialog boxes, see the Microsoft Windows User's Guide.

Dialog boxes and overlay windows appear when you choose certain menu items or click some command buttons. You will know when a command will cause a dialog box to appear because the command will have an ellipsis (...) appended to it.

A dialog box presents a list of options available for the selected command and asks you to type in information that the program requires to proceed. It has buttons at the bottom of the screen (typically OK and Cancel, for example) that cause your selections/entries to be enacted. You cannot proceed to other actions until you close a dialog box.

A window overlay is much like a dialog box except that it does not have buttons and must be closed by clicking in the close box in the upper right corner of the window. Windows do not need to be closed to take other actions in the main window.

Following are some of the typical items in dialog boxes and windows used in the IonWorks HT user manual:

OK Button

The OK button carries through the choices you specify in a dialog box, closes the dialog box, and returns you to the program.

Cancel Button

The Cancel button causes the application to ignore any changes made in the dialog box at that point and returns you to the document window.

Browse Button

The Browse button displays an ellipsis. Clicking it opens the Browse for Folder dialog box which allows you to locate a specific folder directory on your computer.



File Menu Dialog Boxes

Open Data DialogBox

The Open dialog box is displayed when you select Open Data or Import Protocol from the File menu or click the Open toolbar button.

The following table describes of the commands in this dialog box.

Command Description/Function

Look in This drop-down menu and the toolbar buttons next to it allows you to browse your computer hard drives and select a folder.

The contents of the folder is displayed in the field below.

File Name A field that displays the name of the folder selected

Files of Type A drop-down menu that allows you to select the file type, or extension. For IonWorks HT Data files, the file type is *.ihe. For protocol files, the file type is *.ihp.

Open When clicked, opens the file in the main window of the software.

Cancel When clicked, closes the dialog box without opening any file.



Save As DialogBox

This dialog box is displayed when you select Save As or Save Protocol from the File menu or when you click the Save button on the toolbar.

The following table describes of the commands in this dialog box.


Save in A drop-down menu with toolbar buttons next to it which allows you to browse your computer hard drives and select a folder in which to save your data or protocol the file type is *.file.

The contents of the folder is displayed in the field below.

File Name A field that displays the name of the selected folder.

Files of Type A drop-down menu that allows you to select the file type, or extension. For IonWorks HT Data files, the file type is *.ihe. For protocol files, the file type is *.ihp.

Open When clicked, opens the file in the main window of the software.

Cancel When clicked, closes the dialog box without opening any file.



Configure TraceExport Dialog Box

This dialog box appears when you select Export Trace from the File menu. It allows you to export data from multiple wells of an experiment in ASCII text file format for subsequent analysis in a spreadsheet application.

Note Export Trace is only available when a data file is open.

The following table describes the commands in this dialog box.


Export by PatchPlate Wells Labels the exported data using row and column designation used by the PatchPlate.

Export by Compound Plate Wells Labels the exported data using row and column designation used by the Compound Plate.

Wells to Export All in Plate Exports the data for all wells

Wells to Export Along Row... Allows you to export data from defined rows, using the Start from and End with fields.

Wells to Export Along Column... Allows you to export data from defined columns, using the Start from and End with fields.

Data Sets to Export Hole Test When selected, the data from the Hole Test will be exported.

Data Sets to Export Seal Test When selected, the data from the Seal Test will be exported.

Data Sets to Export Pre-compound When selected, the data from the Pre-compound scan will be exported.

Data Sets to Export Post-compound When selected, the data from the Post-compound scan will be exported.

Data Sets to Export Command Voltage When selected, the command voltages will be exported.



Configure MetricsExport Dialog Box

This dialog box appears when you select Export Metrics from the File menu. It gives you options on how you want your exported data to be configured.

Note Export Metrics is only available when a data file is open.


Export File Prefix Allows you to enter a prefix to the file name that is exported.

Browse Allows you to select a file destination.



Export by PatchPlate well Labels the exported data using the row and column designation used by the PatchPlate.

Export by Compound-Plate well

Labels the exported data using the row and column designation as defined in the experimental protocol.

Metrics to Include: User-Defined Only

When checked, the export file will only contain the user-defined metrics shown in the Edit Metrics dialog box.

Metrics to Include: Exclude Hole & Seal-Test

When checked, the results of the hole and seal test will not be included in the exported file.

Select Formatting Options: Export by Row

When selected, the data will be placed horizontally across the page.

Select Formatting Options: Export by Column

When selected, the data will be placed vertically down the page.

Select Formatting Options: Suppress column headings and footer info

When checked, all column headings and footer information will not be exported.

Export Filename Enter a name for the metrics file to be exported.



Experimental Protocol Properties Dialog Boxes

About the ProtocolProperties Dialog

Box

This dialog box appears when you select Edit Protocol from the Edit menu. This is where you will set up all the conditions and parameters of your protocol.

The Experimental Protocol Properties dialog box has four tabs:

♦ Name File—This tab contains file naming and saving options

♦ Start → Seal Test—This tab contains the options and settings for running the seal test on the PatchPlate

♦ Access→End—This tab contains the options and settings for running the patch clamp experiment, after the hole and seal test are performed

Note Some of the Protocol Properties tabs contain additional dialog boxes. These tabs and the dialog boxes they contain are described below.

Name File Tab

The table below describes the items and options in the Name File tab:

Item Description

Select a File Naming Method

This pane allows you to choose an option for naming you data file:

Select... If...

Date Prefix You want your data file name to contain the date the experiment was run.

Custom Prefix You want to create your own prefix.

Type the prefix in the blank field.

Note The following characters are not allowed in the Custom Prefix: \ / : * ? < >

Prompt at Run Time

You want to wait to enter a file name until the experiment is started.



Start —> Seal TestTab

This tab contains options for preparing the PatchPlate (including partial-plate coverage options), parameters to adjust the voltage pulse for the Seal Test performed after the cells are added to the PatchPlate and a place to enter remarks on the experiment.

Clicking this opens the Browse for Folder dialog box and allows you to select a file directory for saving the data files.

Experimental Summary

This pane is present in all the Protocol Properties tabs and contains a summary of all the conditions and parameters that will be executed in the experimental run.

Note When changes are made to the protocol, the Experimental Summary pane will be dimmed until you click the Apply button. Clicking Apply will update the Experimental Summary information.

The table below describes the items and options in the Name File tab: (continued)

Item Description



The table below describes the items and options in the Start Seat Test tab:

Item Description

Displays the steps of the experiment that will be executed chronologically from the start through the seal test and indicates uncompleted, active and completed steps.

Configure Prime Plate In this pane you determine how much of the PatchPlate will be used in the experiment. The choices are:

♦ Whole Plate—When selected, all 384 wells will be used.

♦ 1/2 plate using—When selected, rows A,D,E,F will be used for the first-half and C,D,G,H for the second-half.

♦ 1/4 plate using—When selected, this option will use rows A,E for the first-quarter, B,F for the second-quarter, C,G for the third-quarter and D,H for the fourth-quarter.

♦ 1/8 plate using—When selected, this option will use every other well of rows A,E for the first-eighth starting with well A1, then the un-used wells from rows A,E for the second-eighth starting with well A2; every other well from rows B,F for the third-eighth starting with well B1, then the unused wells from rows B,F for the fourth-eighth, etc.

Add perforation agent during “Prime Plate”

When checked, the plenum will be primed with fluid from the Antibiotic source bottles instead of the Internal Buffer source bottle.

Configure “Seal Test” In this pane you determine the settings for the seal test, i.e. duration and voltage applied to the cells.

Time to Wait Before Seal Test—Type in the length of time (in seconds) you want to wait between the Prime Plate and Start Seal Test (value range = 1–1800 seconds)

Max (mV)—Type in the maximum voltage (in mV) you want applied to the cells during the seal test (value range = -995 to 1000).

Min (mV)—Type in the minimum voltage (in mV) you want applied during the seal test (value range = -1000 to 995).

Period (in msec)—Type in the duration (in sec) for which the max and min voltages should be applied to the cells during the seal test (value range = 40–500).

Configure “Hole Test” This pane allows you to set the limits (in mV) for the hole test.

Max (mV)—Type in the maximum voltage value you want applied during the hole test.

Min (mV)—Type in the minimum voltage value you want applied during the hole test.

Enter Remarks This field is reserved for entering comments.

Prompt on the Run—When selected, you will be asked to enter remarks when you start the experiment.

Enter Now—When selected, the comments field in the current dialog box is enabled and you may enter your comments.



Experiment Summary This pane is present in all the Protocol Properties tabs and contains a summary of all the conditions and parameters that will be executed in the experimental run.


The table below describes the items and options in the Start Seat Test tab: (continued)

Item Description



Access—>End Tab This tab contains options for obtaining and testing electrical access to the cells, selecting the type of experiment to run and provides access to other editing dialogs for the command voltage waveform and the plate-scanning protocol.

The table below describes the items and options in the Access to End tab:

Item Description

Displays the steps of the experiment that will be executed chronologically from the Obtain Access step through the end of the experiment and indicates uncompleted, active and completed steps.



Configure “Obtain Access” In this pane, you determine the operation performed and duration for all desired steps during the Obtain Access portion of the experiment.

The drop-down menu of each step has the following choices:

♦ Introduce Agent—When selected the instrument will introduce the Perforation Agent into the plenum at this point on the Obtain Access sequence. The duration of this operation is fixed.

♦ Introduce Internal—When selected the instrument will add liquid from the Internal Buffer source at this point in the Obtain Access sequence. The duration of this operation is fixed.

♦ Circulate fluid—When selected the instrument will circulate the liquid in the plenum this point in the Obtain Access sequence. The duration of this operation can be set from 1 to 1200 seconds in one-second increments.

♦ Pause—When selected the instrument will pause operations at this point in the Obtain Access sequence. The duration of this operation can be set from 1 to 1200 seconds in one-second increments.

♦ Check seals—When selected the instrument will run an additional Seal Test at this point in the Obtain Access sequence. The duration of this operation is fixed.

♦ Null Operation—When selected, the step will be omitted.

Note When a choice is selected for a step, the Time field adjacent to the step will become enabled if the duration of the step can be changed. Enter the duration (in sec) for which you want that step to be executed.

When Manually set the duration of the “Obtain Access” step to is checked, the Obtain Access step is allowed to overlap with the actual analysis measurements. The duration cannot be set shorter than the end of the last Check Seals operation in the Obtain Access step. All other operations are allowed to overlap with the analysis measurements.

The table below describes the items and options in the Access to End tab: (continued)

Item Description



Configure Measure Currents In this pane, you determine the settings for the portion of the experiment where voltages are applied to the cells and currents are measured.

Under Select Fluid Handling, there are three options:

Edit Command Voltage and Configure Scan Parameters—For a description of these dialog boxes, see “Waveform Editor Dialog Box” on page 4-31.

Experiment Summary This pane is present in all the Protocol Properties tabs and contains a summary of all the conditions and parameters that will be executed in the experimental run.


The table below describes the items and options in the Access to End tab: (continued)

Item Description

Choose... If...

No fluid additions/ Repetitive scans

You want to take repeated measurements without adding anything to the cells

Single compound addition

You want to take a control measurement and compare it to a second measurement after the addition of an agent.



Waveform EditorDialog Box

This dialog box appears when you click Edit Command Voltage in the Configure Measure Currents pane of the Access→End tab.

In this dialog box you determine the start and stop voltages for each step and duration of each step for the measure currents segment of the experiment.

The table below describes the options in the Waveform Editor dialog box:

Item Description

Reference Potentials Click the Holding Potential , Conditioning Potential , or Pre-signal Parameters buttons to change parameters for each respective setting.

Interval Sequence This pane displays the active user-defined command voltage intervals out of the total number possible (10). By default, one interval is shown. For additional intervals, press the Add Interval button.

To delete an interval, set the Interval Duration to zero. The deleted intervals will be removed from the sequence list once the Waveform Editor is closed.

Note Click Refresh Plot to update the plot of the protocol.

Acquisition Sets the data sampling rate in msec/sample. Select one of eight fixed values from the drop-down menu.



Sequence Repetitions Allows a subset of intervals in the voltage protocol to be repeated. The repeating intervals must be in a single contiguous group, which are specified by selecting the Start Interval and End Interval values from the drop-down menus. If the number is greater than 1, enter the amount of time in the Delay before reapplying field (in msec) that you want to wait between repetitions. Repeated intervals are indicated by yellow shading in the graph.

Tip: Set the End Interval value first in order to avoid getting a warning message.

Note Repetitions cannot be combined with the family option.

Interval Setup Click a Pulse Interval Sequence button and then enter the Starting Potential and Ending Potential (in mV) to be applied at each interval as well as the Interval Duration (in msec) for the start and end interval.

Checkmark Use Family and enter the Family Potential (in mV) to use a family of ion currents produced by a set of voltage pulses where a single pulse is set at one voltage for the first cycle and then at a different voltage for the following cycles. Typically, a “family” contains 8-10 “members” where the only difference is the voltage of a single pulse in the Command Voltage sequence. This capability is useful when investigating the current response of the cell as a function of applied voltage and the plot of the maximum current vs. the applied voltage values is called an I/V curve. In the IonWorks HT software, only two members are allowed and only one interval in the Command Voltage sequence can be used for the family.

When the Use Family option is selected, the Family Potential field is enabled to allow the second voltage setting to be entered.

The table below describes the options in the Waveform Editor dialog box: (continued)

Item Description



Conditioning Potentials These are used to precondition the cell prior to applying the Command Voltage protocol. The potentials can be used to, for example, open the channels after compound addition to allow access to the compound and then return to the Holding Potential to rest the cell before the Pre-signal part of the Command Voltage protocol. The Conditioning Potentials are inserted after the Holding Potential and before the Pre-signal part of the Command Voltage protocol. Cell currents are not recorded during the Conditioning Potentials except for the last few milliseconds as needed to establish a signal baseline.

Checkmark Use Conditioning to enable Conditioning Potentials.

1st step Potential (mV)—Value range is -1000 to 10000 mV. Sets the voltage for the first step, typically to allow compound access to channels.

1st step Duration—Value range 1 to 59 seconds, depending on 2nd step. Sets the duration of the first step. The total length of the two steps cannot exceed 60 seconds.

2nd step Potential—Value range -1000 to 1000 mV. Sets the voltage for the second step, typically to allow the cell to return to the resting state. This value is initially set to match the Holding Potential for the experiment.

2nd step Duration—Value range 1 to 59 seconds depending on second step. Sets the duration of the second step. As noted for the first step, the total duration cannot exceed 60 seconds.

Holding Parameters Holding Potential (value range = -1000 to 1000 mV)—Sets the voltage the cell will be held at both before and after the Command Voltage is applied. This parameter is used to allow the cell to enter a “resting state” where all of the ion channels are closed.

Pre-Signal Holding Time (value range = 3–300)—Refers to the period the cell will be held at the Holding Potential before the Command Voltage is applied.

Post-Signal Holding Time (value range = 0–300 sec)—Refers to the period after the Command Voltage sequence has been completed.

Presignal Parameters In this pane, you determine the voltage settings for a measurement prior to the main signal. This feature is typically used to determine the seal resistance of the cell so it can be compensated for, in the main current measurement.

Enter the Time (in msec) for which you want the Initial and the Final voltages applied to the cells. Enter the voltages (in mV) you want applied during these intervals.

The table below describes the options in the Waveform Editor dialog box: (continued)

Item Description



SetSingle-Addition

Screen ParametersDialog Box

This dialog box appears when you click Configure Scan Parameters in the Configure Measure Currents pane of the Access→End tab and you have Single Compound Addition selected.

In this dialog box, you determine the settings for a patch-clamp measurement if you have chosen the single compound addition option under Measure Currents pane

The table below describes the items and options in the Set Single Addition dialog box:

Item Description/Function

Select Compound-Plate Location and Type

In this pane, select a plate (1 or 2) and then a plate type. This identifies the compound plate. The Select Plate Type list contains a set of pre-defined compound plates for use with the IonWorks HT system. Contact Molecular Devices to request additional plate definitions.

Select 96-well subset—Not enabled unless the compound plate-type is a 384-well microplate. It allows you to run a 384-well plate by quadrants.



Select Timing and Scan Parameters

Compound Incubation Time (value range = 1–1200 sec)—Enter the length of time (in sec) that you want the cells incubated with compound before applying the voltage.

Select Scan Type— HAO, QAO and EAO are the options that determine the order in which the cells are scanned. The order affects the length of time that the cells will be exposed to the compound before the Post-Compound scan is performed. If short exposure times are important. EAO or QAO is the preferred scan type because it keeps delay between compound addition and reading small. The drawback is that the overall experiment time is longer. If HAO is selected, the time between the compound addition and scan will on average be longer but the total experiment time will be shorter. The summary table shows the experiment time for the selected scan type.

Tip: Refer to the Minimum Incubation Time in the summary table and use this value for the Compound Incubation Time, in order to avoid being prompted with a warning message.

Offset Voltage Correction In this pane, you may enter an offset correction. Enter the voltages (in mV) you want to subtract in the pre-compound and post-compound fields.

The table below describes the items and options in the Set Single Addition dialog box: (continued)




Pipettor Options Select the number of standard wash cycles (between 1–10) from the drop-down menu for Wash Cycle.

Checkmark Use Solvent Wash to initiate a solvent wash after each row of compounds are dispensed and mixed in the PatchPlate. Specify the number of solvent wash cycles from the drop-down menu.The solvent wash will be followed by the standard washing protocol at the F-head Wash Station. The pipettor will wash at a different row of the solvent plate after each row of the compound plate. The solvent wash plate should have sufficient volume in the wells to exceed the dispensed compound volume. Select the plate type which will contain the solvent from the Plate Settings drop-down menu. The ability to choose the solvent wash plate selection is only possible when the Use Solvent Wash box is checked .The plate type used to hold solvent can be different from the compound-plate type.

Note If a boat-type plate is used, it must be added to the plates.txt file and properly calibrated. The pipettor will move to different positions within a boat-type plate.





Pipettor Options (continued) Select the number of compound mix cycles (between 4–10) from the drop-down menu for Compound Mix Cycle.

Checkmark Use Extra Aspirate Volume when highest pipetting performance is desired (this setting marginally increases experiment time). Checkmark Use Singleton Dispense to transfer compound into each well of the PatchPlate independently, rather than in quadruplicate from a single draw from the compound plate. This option improves the dispensing precision for the four replicate wells. It can be combined with the Extra Aspirate volume option as well, but it should be noted that the extra aspirate volume will be drawn with each transfer, not divided among all four singleton transfers. The Singleton Dispense option adds roughly 2.5 minutes to the total running time of an entire PatchPlate; this is reflected in the Timing and Scan Parameters table.





Analyze Data Tab

The table below describes the items and options in the Analyze Data Tab:

Item Description

Configure Analysis Metrics There are three options:

♦ Click New Metric to open the Design Analysis Metric dialog box and add a new metric.

♦ Select a metric to edit from the Menu of Analysis Metrics and then click Edit Metric, to open the Design Analysis Metric dialog box and edit the selected analysis metric.

♦ Select a metric to delete from the Menu of Analysis Metrics and then click Delete Metric.

Note Refer to page 4-39 for a description of the Design Analysis Metric dialog box options.

Automatic Metric Export When checked, data will be exported automatically. Click Configure Metric Export to open the Configure Export Metric dialog box and define export settings (see page 4-23 for a description of this dialog box).

Note If you choose to enter a filename and not use the auto-naming option, and the filename already exists in the directory, a warning message will be displayed. When this occurs, you can click OK and then export the file manually.

Automatic Trace Export When checked, trace data will be exported automatically. Click Configure Trace Export to open the Configure Export Trace dialog box and define trace export settings (see page 4-22 for a description of this dialog box).

Note If you choose to enter a filename and not use the auto-naming option and the filename already exists in the directory, a warning message will be displayed. When this occurs, you can click OK and then export the file manually.



Design AnalysisMetric Dialog Box

This dialog box appears when you click New Metric or Edit Metric in the Analyze Data tab of the Experimental Protocol Properties dialog box. These computations are analysis metrics performed on the measurements taken during the experiment. Designing a new metric is performed in four steps.



Note The maximum number of metrics that can be created is 20.

Below is a description of the items and options in the Design Metrics dialog box:

Item Description

Chart Controls A chart of the Command Voltage graph can be used to assist in identifying time or voltage settings for analysis metrics.

Zoom—To select a zoom mode, press the Horizontal Zoom , Vertical Zoom or Four-Way Zoom button to

enlarge a region of the graph. Then zoom on a region of interest by click and dragging. Press the Undo Zoom button

to reset to the un-zoomed view. Position the pointer anywhere on the chart to read the time and voltage measurements for that position. The value is displayed below the chart, entitled Cursor Time-Voltage Value.

Press the left mouse button anywhere on the chart and a red vertical line with the time value will be automatically graphed.

Step1:Label for the Metric Type in or edit a name in the field.

Step 2:Select the Metric Type

Choose one, either a simple metric or a combination metric. These are defined in Step 3.



Step 3: Design the Metric The graphic shows a plot of the Command Voltage i.e., the voltage protocol as defined in the Waveform Editor dialog box. This allows you to set regions of interest (start and end times) based on when certain pulses will be applied to the cell.

The metric definitions are applied to the set of current measurements obtained from each well of the PatchPlate, not across all wells. The set may be the entire set measured during the scan or it may be only the data points that fall into the time interval set by the user.

From the appropriate Metric drop-down menu choose one of the following metric definitions:

♦ Minimum—Uses the minimum value determined from the data acquired for each individual cell during each scan.

♦ Maximum—Uses the maximum value determined from the data acquired for each individual cell during each scan.

♦ Mean—Uses the mean value determined from the data acquired for each individual cell during each scan.

♦ Absolute Value of Min—Uses the absolute minimum value determined from the data acquired for each individual cell during each scan.

♦ Absolute Value of Max—Uses the absolute maximum value determined from the data acquired for each individual cell during each scan.

♦ Absolute Value of Mean—Uses the absolute mean value determined from the data acquired for each individual cell during each scan.

♦ Standard Deviation—Uses the standard deviation value determined from the data acquired for each cell during each scan.

♦ Maximum Signal Time—Uses the time at which the maximum (peak) current is measured.

♦ Minimum Signal Time—Uses the time at which the minimum current is measured.

After choosing the metric, choose a time interval for evaluation of the metric. Choose one of the following:

♦ Select Entire Signal to evaluate the entire duration of the applied signal.

♦ Select From to to evaluate a portion of the signal and enter the desired start and stop time points (in msec).

Below is a description of the items and options in the Design Metrics dialog

Item Description



Edit Metrics MenuDialog Box

This dialog box appears when you select Metrics from the Edit menu in the main window. It enables you to directly access the Design Analysis Metric dialog box (described above) without having to open the Edit Protocol Properties dialog box.

Click New Metric or select a metric form the metric menu and click Edit Metric to open the Design Analysis Metric dialog box.

Select Combination Method If you chose a Combined Metric, then you will also need to select a method of comparison. The metrics chosen in Step 3 are used for the combined method calculation.

Note The Combined Metric option allows you to select two regions of interest in the scan and compare them to each other. As an example, to select one region as a baseline measurement of the ion current and the other as the current when the ion channels should be fully opened during the same scan.

Step 4: Configure the Cross-Family comparison method (Command-Voltage Families Only)

These options are enabled when Families has been selected in the Waveform Editor (see “Waveform Editor Dialog Box” on page 4-31).

Choose a Final Family Metric.

Note The Families option is similar to a Combined Metric. The difference is that a voltage protocol that uses a family produces two scans per well so the comparison is done between these two scans. Therefore, you can create a Combined Metric that applies to a voltage family.

Below is a description of the items and options in the Design Metrics dialog

Item Description



Edit Filter DialogBox

This dialog box appears when you select Filter...> User-Defined from the Edit menu or click Edit Filter in the Configure Filter Metric in the main window. This dialog box allows you to configure new filter metrics or edit the ones already configured.

The items in this dialog box are described in the table below.


Select Filter Enter the filter metric you want to create or modify.

Label Enter the name of the filter metric

Single-Scan Filter Indicates which segment of the experiment to which the metrics will be applied. The scans to choose from will always include the Hole Test and Seal Test, but depending on the type of experiment, there may be more or less than two additional scans. For the Single Compound Addition, there will be two scans labeled Pre-compound and Post-compound, but if the Interactive Scan mode is used, there can be one or more scans labeled Scan #1 etc.

Filter Metric The measurement criterion to be applied to the scans in the experiment. This list will contain any metrics you create using the Edit Metrics dialog box. The Resistance and Offset Voltage metrics are present by default in all protocols. Two examples of User-defined Metrics could be Global Mean, kv Metric.



Filter out wells with metrics that are less/greater than this value.

Allows you to define the exclusion limits for a cell (or well).

Note When determining a threshold value for use with the filter, be aware that the values displayed in the Plate Legend and Statistics Area are rounded to three decimal places, whereas the values used by the Hits Display are much higher precision. Values greater than three decimal places can be specified for the hits threshold.

Update Value When clicked, the settings shown will be applied to the data and the recalculated values will be displayed in the PatchPlate.

Two-Scan Filter Indicates which segment of the experiment to which the metrics will be applied for Scan A and Scan B. The scans to choose from will always include the Hole Test and Seal Test, but depending on the type of experiment, there may be more or less than two additional scans. For the Single Compound Addition, there will be two scans labeled Pre-compound and Post-compound, but if the Interactive Scan mode is used, there can be one or more scans labeled Scan #1 etc.

Filter Metric The measurement criterion to be applied to the scans in the experiment. This list will contain any metrics you create using the Edit Metrics dialog box. The Resistance and Offset Voltage metrics are present by default in all protocols. Two examples of User-defined Metrics could be Global Mean, kv Metric.

Cross-Scan Comparison Method The mathematical relationship used to compare Scan A and Scan B. The default choices are:

♦ % Difference: 100*(Scan B - Scan A)/Scan A

♦ Absolute Value of % Difference

♦ Difference: Scan B - Scan A

♦ Absolute Value of Difference

♦ Ratio: Scan B/Scan A

♦ Absolute Value of Ratio

Filter out wells with metrics that are less/greater than this value

Allows you to define the exclusion limits for a cell (or well).

Note When determining a threshold value for use with the filter, be aware that the values displayed in the Plate Legend and Statistics Area are rounded to three decimal places, whereas the values used by the Hits Display are much higher precision. Values greater than three decimal places can be specified for the hits threshold.




Native Filter DialogBox

This dialog box appears when Filter...Native is selected from the Edit menu. This dialog box allows you to change the resistance acceptance range for the PatchPlate Seal Test. The resistance acceptance range for the PatchPlate Hole Test is not adjustable. These values are provided for reference.

Update Value When clicked, the settings shown will be applied to the data and the results will be updated in the PatchPlate or Compound Plate display.




View Menu Dialog Boxes and Windows

View ActiveProtocol

This dialog box appears when you select Active Protocol from the View menu in the main window. It displays a summary of the current experimental protocol. To modify the current protocol, you may access the Edit Protocol dialog box from the Summary of Active Protocol by selecting Protocol from the Edit menu in the dialog box.

File Menu

The file menu of the Summary of Active Protocol contains the following items:

Item Description

Save Protocol Opens the Save As dialog box and allows you to name, designate a directory and save the protocol as a *.ihp file which can be opened and used again.

Print Opens a Print dialog box which allows you to choose a printer and print the summary.

Exit Closes the Summary of Active Protocol dialog box.



Edit Menu

ExperimentSummary

This dialog box appears when you select Experiment Summary from the View menu in the main window. It contains a record of the parameters and conditions under which the experiment was carried out.

You can save, print and exit the summary using the commands in the File menu.

The Edit menu of the Summary of Active Protocol contains the following items:

Item Description

Protocol Opens the Edit Protocol Properties dialog box so you may edit the current active protocol.



Trace Groups This window appears when you select Trace Groups from the View menu in the main window. This window is only available when there is a data file open.

Trace Groups display a graphic plot of the recorded signals from each cell well for each segment of the experiment i.e., the Hole Test, Seal Test, Scan #1 and Scan #2.

Below is a trace from a Hole Test.

Name of test displayed and date test was recorded

Well group currently displayed

Scan currently displayed

Trace color for A2

Cell location on PatchPlate

Y-axis, displayed in µAmps

X axis displayed in msec

Trace for cell located in A2



Below is a description of the items and options for the Trace Groups:


Drop-down menu that allows you to choose a particular signal trace for viewing.

Indicates and controls which group of wells are currently being displayed. Click the up and down arrows to scroll through all the well traces.



Selecting Scaling opens the Scale All Axes dialog box.

Show Annotations When this option is selected, the cell resistance is shown in the lower-right plot area for each trace. The resistance value is determined using the default resistance metric.

Refresh When selected, the plots are redrawn using the current settings.

Below is a description of the items and options for the Trace Groups: (continued)


Item Description

SealTest and Hole Test Plot Scaling

Choose the method by which you want to scale the tracing for the Seal and Hole Test.

The Fixed Scale with Auto Bias Adjust option plots all traces with the same y-axis range. In this case, the axis range is scales to match the greatest data range.

The Auto Scale option adjusts the y-axis of each plot to match the range of the data.

Measure Currents Plot Scaling

Choose the method by which you want to scale the tracing for Scans 1 and 2.

If you select manual, then type a value into the scale limit fields.



IonWorksMessages Dialog

Box

This dialog box appears when you select Messages and Logging from the View menu. When the IonWorks HT instrument is ready to run experiments, messages between the host PC and the instrument will be captured in this dialog. Messages that appear are not automatically saved.

Note Communications messages will not appear in the IonWorks Messages dialog box until the instrument is connected and powered on.



Receive detailed messages This option is only enabled when the instrument is communicating with the host PC. The detailed messages provide additional information about the actions being taken by the instrument during the experiment.

Note This option should only be used for instrument service purposes and should not be used during regular operation as it will affect the timing of instrument operations.

Start Logging When clicked, the Save As dialog box will open prompting you to name the file and determine the destination of the saved file. The saved logging file will have a *.log file extension.

Stop Logging When clicked, the logging file will stop collecting log and messaging data.


5
Operating the System 5
Overview

In This Chapter This chapter provides information and procedures required to operate the IonWorks™ HT system and run an experiment.

Topic See Page

Overview of Running an Experiment 5-2

Setting Up the IonWorks HT System for an Experimental Run 5-3

Preparing the Cells 5-4

Preparing Buffer Solutions 5-5

Preparing the Perforation Agent 5-7

Preparing Compound Plates 5-8

Preparing the Fluidics System 5-9

Preparing the Experiment Stage 5-11

Setting-Up the Experiment Protocol 5-12

Completing the Experiment Setup 5-14

Exporting Data 5-18

Importing a Data File into Microsoft Excel 5-21

Exporting Metrics 5-23

Importing a Metrics File into Microsoft Excel® 5-25

Shutting Down the System After Completing All Daily Experiments 5-27


Chapter 5 Operating the System

Overview of Running an Experiment

ExperimentOperations

Overview

After issuing the start command for running an experiment, the following operations are executed:

1 12-channel pipettor aspirates the appropriate solution from the Buffer Reservoir station on the experiment stage and performs multiple dispenses into the wells of the PatchPlate.

2 The fluidics panel primes the lines that feed the plenum or lower chamber under the PatchPlate.

3 48-channel electronics head (E-head) records the electrical current flow through the small “holes” at the bottom of the PatchPlate (a.k.a. the Hole Test).

4 The pipettor adds cells from the Cell Suspension Reservoir to the PatchPlate. At this point, there is a waiting period (user-determined) for cells to reach and to seal the holes at the bottom of the PatchPlate.

5 The E-head performs another recording to check the status of the cell’s seal formation (a.k.a. the Seal Test).

6 The perforation agent is introduced in the lines that feed the plenum or lower chamber under the PatchPlate. The perforation agent is cycled through the plenum to optimize access to the inside of the cells.

7 48-channel E-head applies the user-defined signals to individual wells and records the ion-channel activity (a.k.a Pre-Compound Scan or Scan 1)

8 The pipettor adds compounds to the PatchPlate from the appropriate compound plate positions. At this point, there is a waiting period for compound incubation.

9 48-channel E-head performs electrical recordings of the PatchPlate to gather data after the compound addition (a.k.a. Post-Compound Scan or Scan 2).

10 The IonWorks HT system performs a cleanup sequence to drain all the fluid lines and to wash both the pipettor and the E-head.


Setting Up the IonWorks HT System for an Experimental Run

Setting Up the IonWorks HT System for an Experimental Run

About the SettingUp the System

Setting up the system requires performing the following tasks:

♦ Preparing cells

♦ Preparing buffer and perforation agents

♦ Preparing compound plates

♦ Preparing the liquid-delivery system

♦ Preparing the experiment stage

♦ Setting up the experiment protocol

♦ Completing the experiment setup



Preparing the Cells

Cell Densities The recommended cell density for running an experiment is ~1 x 106 cells / mL. While growth patterns vary widely for each cell-line, cells must reach 70-100% confluence levels for optimum ion-channel expression. Based on their growth cycle, cells must be sub-cultured in advance to allow for 70-100% confluency on the day of the experiment. The cells are loaded into v-bottom removable reservoirs for pipetting into the PatchPlate. The minimum required volume for the final cell suspension should be a minimum of 2.5 mL.

Cell PreparationRequirements

Cells are removed from their growth media and are suspended in balanced salt solutions for preparing the final cell suspension. The final cell suspension that is pipetted into the PatchPlate should contain evenly distributed cells with no clumping. Cell preparation techniques should provide a final cell suspension with isolated single cells. The preparation methods for disassociating cells should neither harm the cell membrane nor affect the ion-channel expression. The gentle mixing action of a standard hand-pipettor (e.g., 200 µL capacity) has proved the preferred method. The number of aspirating and dispensing strokes should be optimized for each cell line. The speed and forcefulness of strokes should be adjusted so as to mix the cell suspension gently.

Window ofOpportunity

The “cell clock” starts at the instant when cells are removed from their growth media. According to this clock, the window of opportunity for recording stable electrophysiological response is limited. Within this time frame, all preparation steps must be planned, scheduled, and executed to optimize the experiment performance. Most preparation techniques require spinning cells in a centrifuge for removing cell-growth media. It is recommended to centrifuge the cells at 50 x g for ~4 min. Centrifugation disturbs the cell’s normal activities. On the other hand, overall stability of cell response is jeopardized when cell recordings fall outside the one-hour window of opportunity that is defined by the “cell clock”.


Preparing Buffer Solutions

Preparing Buffer Solutions

About BufferSolutions

The balanced salt solutions are used to maintain cells for a short time in a viable condition rather than to promote their growth. During this time, the cells need to be maintained in a medium of physiological pH and osmotic balance. The choice and formulation of these buffer solutions depend on the cell line and the ion channels of interest. The IonWorks HT system uses both extracellular (i.e., External Buffer) and intracellular solutions (i.e., Internal Buffer) for maintaining and optimizing the cell’s electrochemical gradient during electrophysiological recordings.

External Buffer The external buffer is equivalent to the “bath” solution in a patch-clamp configuration. External buffer has the following applications in the IonWorks HT system:

♦ Preparation of cell suspension

♦ Filling the PatchPlate

♦ Washing solution

The recommended external buffer for use in IonWorks HT system is the standard Dulbecco’s Phosphate Buffered Saline solution (1X) with calcium and magnesium (filtered with a 0.1 µm filter).

IMPORTANT Any solution used for pipetting into the PatchPlate must be both sterile and 0.1 µm filtered.

Internal Buffer The internal buffer is equivalent to the “pipette” solution in a patch-clamp configuration. Internal buffer has the following applications in the IonWorks HT system:

♦ Priming the plenum lines at the beginning of the experiment

♦ Filling the plenum line during the experiment

♦ Dissolving the perforation agent

The choice and the composition of the internal buffer depend both on the host cell and the ion channel of interest.

Note While internal solutions may be optimized for the ion channel of interest, they must maintain the required physiological pH and osmotic balance for the host cell at all times. In essence, the internal solution composition must be optimized for preventing washout of cytosolic constituents following patch excision or dialysis of cells during long-lasting whole-cell measurements.

DeterminingVoltage-Offsets

IMPORTANT Improper voltage-offsets will result in absolute voltage errors and suboptimal performance.

Various combinations of external and internal solutions cause liquid-junction potentials in the IonWorks HT system. Liquid-junction potentials (defined as potential differences between two solutions) occur at all boundaries between solutions of different compositions. The IonWorks HT system approximates the voltage-offsets that arise from liquid-junction potentials during the first portion of the experiment prior to the cell addition (i.e., during the Hole Test). The final correction factor that is applied to compensate for these voltage-offsets during cell recordings, however, require a user-input value for the actual voltage-offset. The voltage-offset values must be determined empirically for each combination of external and internal solutions. A



biological indicator, such as the time-profile response of an ion channel, can be used to determine the voltage-offsets. For example, one biological indicator is the potassium voltage-gated ion channel (Kv1.52e6). The shift in the inflection point in response to a “ramp” command voltage can be used to empirically determine both the magnitude and the sign (i.e., positive or negative) of the voltage offsets due to liquid-junction potentials.


Preparing the Perforation Agent

Preparing the Perforation Agent

About PerforatingAgents

The IonWorks HT system uses a perforation agent that forms low-resistance pathways through the cell membrane for gaining “access” to the inside of the cell. The low-resistance access allows the passage of only small ions and, thereby, prevents the washout of cytosolic constituents. Amphotericin B, a polyene antibiotic with an anti-fungal profile is the recommended perforation agent for use in the IonWorks HT system.

UsingAmphotericin B

Amphotericin B in powder form is dissolved in dimethylsulfoxide (DMSO) and diluted in the internal buffer. Amphotericin B is a poorly soluble compound. Therefore, it is crucial to dissolve the Amphotericin B in DMSO before diluting the solution in the internal buffer. A recommended method is to increase the surface area of the Amphotericin B in powder form when adding the DMSO. The final Amphotericin B solution must be uniformly clear and devoid of any precipitates.

Note The final DMSO concentration should not exceed 0.5%.

Note The recommended method for use of Amphotericin B is to prepare the solution fresh each time prior to the start of an experiment.



Preparing Compound Plates

When to Preparethe Compound

Plate

Depending on its complexity, preparing the compound plate can take a variable amount of time. For this reason, it is best to plan your experiment carefully to ensure that the compound plate will be ready for use as soon as the cells are ready to be assayed.

Compound PlateTypes

Either 96- or 384-well microplates can be used in the compound plate positions of the experiment stage in the IonWorks HT system. You must specify the plate-type from a list provided in the system software prior to instrument operation.

Dispensing fromCompound Plates

Each well of the compound plate (either 96- or 384-well) supplies four wells of the PatchPlate with the compound solution.

96-Well Plate

A 96-well compound plate is mapped to the 384-well PatchPlate such that there are four corresponding PatchPlate wells for each compound-plate well.

384-Well Plate

A 384-well compound plate is regarded as four separate 96-well compound plates where a 96-well subset of a 384-well compound plate is used in an experiment. The 96-well subset consists of every other well in a 384-well compound plate which is specified through the system software.

Quantity of Fluid Dispensed

The fluid dispensed into the PatchPlate well from the compound plates is done in 3.5 µL aliquots. For each experiment, the required pipetting volume for loading the PatchPlate with the compound solution is 14 µL (4 x 3.5 µL) per compound-plate well. The required fluid volume in each well of the compound plate must also account for the dead-volume due to pipetting heights. For example, the dead-volume for a 1/2 area 96-well plate (Costar P/N 3696) is approximately 35 µL. Therefore, a minimum fluid volume should be 49 µL (14 µL + 35 µL) for each well of the 96-well plate.

Concentration ofCompounds in the

Plate

The compounds must be prepared at 3X the final concentration. When added to the PatchPlate, the compounds are diluted 1:3 from their original concentration in the compound plate. Prior to compound addition, each well of the PatchPlate is filled with 7.0 µL of fluid (3.5 µL of external buffer and 3.5 µL of cell suspension). The compound addition of 3.5 µL diluted the compound concentration by a factor of 1:3 (3.5:10.5 µL).


Preparing the Fluidics System

Preparing the Fluidics System

Preparing theLiquid-Delivery

System

Note When starting an experiment, the source containers should be clean (refer to “Shutting Down the System After Completing All Daily Experiments” on page 5-27 for a procedure to clean the system after an experiment).

To prepare the liquid-delivery system:

Step Action

1 Perform the system start-up procedure (see “Starting-Up the IonWorks HT System” on page 3-2).

2 Drain all waste bottles.

IMPORTANT Turn the bottle to unscrew the bottle cap when connecting or removing the bottles. All bottle caps are connected to internal tubing lines and turning the bottle caps instead of the bottles will introduce kinks into the internal tubing lines.

IMPORTANT Check that all bottle caps are tightly secured prior to the start of an experiment. The waste bottle caps are connected to the main system vacuum line and must be tightened to create a closed vacuum system. Loose bottle caps will results in vacuum leaks and improper priming of the PatchPlate, Avoid exerting excess force in tightening the bottle caps as over-tightening can damage the locking threads on both the cap and the bottle.

CAUTION Always check the fluid volume in the waste bottles prior to starting an experiment to avoid overflowing and saturating the vacuum systems. The waste-bottle caps are connected to the main system vacuum line. Overflow of the waste bottles will damage the vacuum system.

3 Drain all source bottles.

IMPORTANT Turn the bottle to unscrew the bottle cap when connecting or removing the bottles. All bottle caps are connected to internal tubing lines and turning the bottle caps instead of the bottles will introduce kinks into the internal tubing lines.

4 Rinse source bottles thoroughly with clean, filtered water.

5 Fill the Antibiotic source bottle with ~150 mL of Internal buffer.

6 Fill the Internal Buffer source bottle with internal (i.e., “pipette” buffer).

Note Each experimental run uses ~100 mL of the internal solution from the Internal Buffer bottle. Adjust the final volume used in the Internal Buffer bottle based on the total number of intended runs. Remember to account for the dead-volume in each bottle.

7 Fill the Electrode Wash and Pipettor Wash source bottles with appropriate wash solution. The recommended wash solution is Dulbeccos Phosphate Buffered Saline 1X solution.

Note Each experiment run uses ~100 mL of the wash solution in each wash bottle. Adjust the final volume of the wash bottles based on the total number of intended experimental runs. Remember to account for the dead-volume in each bottle.



8 Load all source bottles.

Note Check that the label on the bottle cap matches the labeling on the appropriate source bottle. The bottle cap contains the line that connects to the appropriate station. The bottle cap is distinguished by a label that corresponds to its matching bottle. Installing the incorrect bottle cap to the source bottle will result in flow of incorrect solution to various stations.

IMPORTANT Check that all bottles are seated properly in the holding rack positions. All bottles contain a flange at the lower part of the bottle neck. This flange supports the weight of the bottle as it is inserted in the holding rack. Improper seating of the bottles will damage the bottles and the connected tubing.

9 From the Utilities menu, select Flush and Rinse. Repeat this command a second time if there is residual ethanol from previous cleaning.

To prepare the liquid-delivery system: (continued)

Step Action


Preparing the Experiment Stage

Preparing the Experiment Stage

About theExperiment Stage

The experiment stage is the area on the instrument surface that contains the PatchPlate, plenum, reservoirs for cells, compound plates, and wash solutions.

Loading aPatchPlate

Preparing theFluid Lines and

12-ChannelPipettor

To load the PatchPlate:

Step Action

1 Open the instrument access door.

2 Check that all stations with removable reservoirs and compound plates are empty.

3 Load an old PatchPlate into the PatchPlate station.

a. Check, and if necessary, dry any traces of fluid on the top surface of the PatchPlate plenum.

CAUTION Keep the top surface of the plenum dry at all times. The top surface of the plenum houses the vacuum port. Avoid introducing any moisture into the vacuum port as moisture will damage the vacuum pump.

b. Place the PatchPlate into the plenum cavity.

c. Seat the clamping frame on the PatchPlate station.

d. Turn the PatchPlate vacuum switch from Release to Hold.

e. Apply an even force to the clamping frame to create the vacuum seal.

Note The vacuum-seal formation is complete when vacuum air leakage is no longer audible at the clamping-frame interface.

To prepare the 12-channel pipettor:

Step Action

1 Clean and dry removable v-bottom reservoirs.

2 Insert one v-bottom reservoir into the Buffer station on the experiment stage.

CAUTION Check and verify that the removable reservoirs are fully seated in the appropriate station on the experiment stage prior to the start of the experiment. Partial seating of the reservoir will damage the E-head and /or fluidics head.

3 Insert a flat-bottom reservoir into the electrode-head (E-head) wash station.

CAUTION Do not confuse this reservoir with the v-bottom reservoir. Inserting a v-bottom reservoir in the E-head wash station will damage the E-head.

4 Fill the v-bottom reservoir in the Buffer station with 5 mL of PBS.

IMPORTANT Make sure that the solution dispensed into the reservoir is sterile and filtered through a 0.1 µm filter.

5 Fill the flat-bottom reservoir with 10 mL of PBS.

6 Close the access door and turn the handle to the lock position.

7 When the experiment start button is activated, indicated by a green traffic light on the main window toolbar, select Flush and Rinse from the Utilities menu.

The system will perform a flush and rinse cycle to clean the fluid lines and 12-channel pipettor. When the cycle is completed, the start button will turn green.

8 After completion of the flush and rinse cycle, open the access door, discard the contents of the v- and flat-bottom reservoirs, clean them, and reinstall them.



Setting-Up the Experiment Protocol

Loading andModifying the

Default Protocol

To set -up the experimental protocol:

Step Action

1 In the main window of the software, select Active Protocol located under the View menu.

2 Check the experiment settings in the Summary of Active Protocol window

If you need to change a setting, close the window and then select Edit Protocol located under the Edit menu.


Setting-Up the Experiment Protocol

3 When the protocol settings are correct, close the Summary of Active Protocol window and start the experiment.

Note For a description of protocol options and settings, see Chapter 4, “System Software Description.”

To set -up the experimental protocol: (continued)

Step Action



Completing the Experiment Setup

Prepare and Loadthe Perforation

Agent

Prepare the perforation agent as described in “Preparing the Perforation Agent” on page 5-7 and replace the contents of the Antibiotics source bottle with the perforation solution.

Check Solutionsand Stations

Check to be sure that:

♦ All source bottles are filled with the correct solution types

♦ All source bottles are filled with sufficient solution volumes

♦ All stations on the experiment stage with removable reservoirs and compound plates are empty

Preparing andLoading the

Compound Plates

Preparing andLoading the Cells

To prepare and load the compound plates:

Step Action

1 Prepare the compound plate.

CAUTION Check and verify that the system software is configured for the correct plate (96- or 384-well) type and the corresponding compound plate station (plate 1 or plate 2). Failure to configure the software or to old the correct plate type will damage the fluidics head.

2 Load the prepared compound plate into the correct compound plate position.

CAUTION Check and verify that the compound plate is loaded in the correct position, fully seated in the experiment stage, and all plate lids have been removed.

The following reagents and supplies are required to prepare the cells:

Item Source

Versene GIBCO P/N 15040-066

Media GIBCO DPBS P/N 14040

15-mL sterile, conical centrifuge tubes

Major laboratory supplier

To prepare and load the cells:

Step Action

1 Remove the media and rinse a T-75 flask of cells (grown to 60-90% confluence) with 5 mL of Versene (at 37 °C).

2 Add 3 mL of Versene and incubate flask at 37 °C for 7–9 min or until the cells become rounded as viewed under a microscope.

3 Tap cells loose and add 20 mL of serum-free cell media to the flask.

4 Decant the cell suspension into two 15- mL conical centrifuge tubes and pellet the cells (50 x g for 4 min).



Loading a NewPatchPlate

CAUTION Keep the top surface of the plenum dry at all times. The top surface of the plenum houses the vacuum port and the presence of moisture will damage the vacuum pump.

5 Add 2.5–3.5 mL (or enough to achieve a final concentration of cells at 1 x 106 cells/mL) of serum-free cell media and resuspend the cells carefully titurating them slowly with a pipettor set at 170 µL.

IMPORTANT It is important to resuspend the cells carefully to avoid clumping.

Note To ensure proper functioning of the system, make sure that the final volume of cells is no less than 2.5 and no more than 3.5 mL.

6 Make sure there is a clean and dry v-bottom reservoir in the Cells station.

CAUTION Check and verify that the reservoirs are fully seated in the experiment stage. Partial seating of the reservoir will result in damage to the electronics and/or fluidics head.

Pour the cell suspension into the cell boat on the experiment stage.

IMPORTANT Check and verify that the cell suspension is evenly distributed in the v-bottom reservoir. Due to fluid surface tension, the cell suspension may collect at one end of the reservoir upon addition. The cell suspension must be evenly distributed in the v-bottom reservoir to ensure proper cell pipetting into the PatchPlate by all 12-channels of the fluidics head.

7 Install a clean and dry v-bottom reservoir into the Buffer station on the experiment stage.

CAUTION Check and verify that the reservoirs are fully seated in the experiment stage. Partial seating of the reservoir will result in damage to the electronics and/or fluidics head.

Dispense 5 mL of external buffer into the reservoir and 10 mL into the E-head wash boat.

8 Install a clean and dry flat-bottom reservoir in the E-head Wash/Soak station and dispense 10 mL of external buffer into the reservoir.

9 Close and secure the instrument cover.

To prepare and load the cells: (continued)

Step Action

To load a new PatchPlate:

Step Action

1 Check and if necessary, unload the old PatchPlate from the PatchPlate station on the experiment stage:

a. Turn the PatchPlate vacuum switch to the Release position.

b. Remove the clamping frame.

c. Remove the PatchPlate from the PatchPlate plenum.

2 Check and if necessary, dry any traces of fluid on the top surface of the PatchPlate plenum.

3 Place the new PatchPlate into the plenum cavity.

4 Seat the clamping frame on the PatchPlate station.

5 Turn the PatchPlate vacuum switch from the Release to the Hold position.



Starting theExperiment

Viewing theExperiment Data

After starting the run by clicking the green button in the main window toolbar or selecting Run from the Experiment menu, the Trace Groups window will open and display the signals in real-time while the software is recording and saving the data.

The experiment segment being recorded is shown in the Scan field and the current group of wells for which the signal is being recorded is displayed in the Group field.

When the run is completed, the Trace Groups window will close and the Data file displaying the results of the run will appear in the main window.

For a description of the Data File window see Chapter 4, “System Software Description.”

6 Apply an even force to the clamping frame to create a vacuum seal.

Note The vacuum seal is complete when vacuum air leakage is no longer audible at the clamping frame interface.

To load a new PatchPlate: (continued)

Step Action

To start the experiment:

Step Action

1 Close the instrument access door and turn the handle to the lock position.

2 Wait for the traffic light icon on the main window toolbar of the system software to turn green.

3 Click the green-lighted button or select Start from the Experiment menu to start the experiment run.



Post-ExperimentOperations

Wait for the experiment run to complete all its operations. Completion of all operations is indicated by the following:

♦ The experiment timer will finish its count-down.

♦ The primary analysis window will display the PatchPlate view along with a summary of the experiment data.

♦ The traffic light icon on the main window toolbar of the system software will turn green indicating the experiment is completed and the software is ready for the next run.

When the experiment is completed:

Step Action


2 Remove the v-bottom removable reservoirs from the Cells and Buffer stations on the experiment stage.

3 Discard the contents and clean the removable reservoirs.

IMPORTANT If reusing, the v-bottom reservoirs should be cleaned thoroughly so that the risk of contaminating the next experimental run is minimized

4 If necessary, remove the compound plates from the experiment stage.

5 Turn the PatchPlate vacuum switch to the Release position.

IMPORTANT Leave an old PatchPlate in the plenum during the instrument “idle” times to prevent ambient debris from entering the plenum and plenum-tubing lines.

6 Continue with the post-run analysis from the primary experiment window. See “Exporting Metrics” on page 5-23 for a procedure on how to export a data file.



Exporting Data

About ExportingData

Data signals recorded during a run may be exported in two ways:

♦ Right-clicking on a signal chart of an individual well and selecting Export Dialog

♦ Selecting Export Data from the File menu and exporting data from multiple wells at the same time

In the first case, the data may be exported in multiple formats, and in the second case, the data is exported in a tab-delimited format, suitable for importing into a spreadsheet application such as Microsoft Excel®.

Exporting Data fora Single Well

To export data from a single well:

Step Action

1 Double-click on the well of interest, in either the pre- or post-compound plate. This opens the data chart for that well.


Exporting Data

2 Right-click on the chart and select Export Dialog form the menu.

3 In the Export pane, select the desired export format.

4 In the Export Destination pane, select the desired output destination.

5 In the Object Size pane, specify the size of the file, if outputting it as a picture.

6 When you have selected all the appropriate options, click Export.

If you select File as the Export Destination, click Browse and navigate to the desired directory for saving the file.

To export data from a single well: (continued)

Step Action



Exporting Data forMultiple Wells

To export the data for multiple wells in tab-delimited format:

Step Action

1 Open the data file from which you want to export.

2 Select Export Data form the File menu. This opens the Configure Data Export dialog box.

3 In the Export By and Wells to Export panes, select the wells from which you want to export the data.

Note Wells exported when using the Along Row option will be different than for the Along Column option if the selected wells span more than one row or column. The exported wells “wrap around” to the next row or column as shown in the following example:

Along Row: from a3 to a16Files: a3,a4,a5,a6,a7,a8,a9,a10,a11,a12,a13,a14.a15,a16

Along Column: from a3 to a6Files: a3,b3,c3,d3,e3,f3,g3,h3,a4,b4,c4,d4,e4,f4,g4,h4,a5,b5,c5,d5,e5,f5,g5,h5,a6,...,a16

4 In the Data Sets to Export, select the data reads you want to export. To select more than one data set, hold the CTRL key down, while selecting the desired data set in the list.

5 To customize the filename prefix, edit the text in the Export File Prefix field. For PatchPlate wells, the software will append ‘_RCC_PP.ihd’ where R is the row letter and CC is the column number. For Compound Plate wells, the software will append ‘_RCC_CP.ihd’.

6 To change the file destination, click Browse to open the Browse for Folder dialog box. Navigate to the desired directory and click OK.

7 When finished, click OK in the Configure Data Export dialog box. A message will appear while the data is being processed to the tab-delimited format.


Importing a Data File into Microsoft Excel

Importing a Data File into Microsoft Excel

About Importing Data from single or multiple wells may be imported into a spreadsheet application such as Microsoft Excel®, if it has been exported in a tab-delimited format. Opening the data file within Microsoft Excel, will launch the File Import Wizard which will guide you through the process.

Importing DataFiles

To import data files into Microsoft Excel:

Step Action

1 Open Microsoft Excel application and select Open from the File menu.

2 In the Open File dialog box, navigate to the data file location.

Note Make sure that All Files (*,*) in the Files of Type field is selected

3 Select the file or files that you wish to open.

Each file comprises the data from one well. To view the data from multiple wells, hold the CTRL key down while selecting the desired files.

4 When finished selecting the files you wish to open, click Open. This will launch the three-step File Import Wizard.

5 Accept the default settings by clicking Next and Finish.



6 Each data set will appear as a pair of columns of time and current or voltage. In the example below, the data from pre- and post compound for well A1 was imported into Microsoft Excel, using the default settings of the File Import Wizard.

PatchPlate Data Import

Compound Plate Data Import

To import data files into Microsoft Excel: (continued)

Step Action


Exporting Metrics

Exporting Metrics

About ExportingMetrics

When exporting the metrics from an experiment, the values obtained for all wells in the plate for each metric defined in the experiment protocol is exported.

If the automatic data export function is enabled, then the data file would already be exported to your designated file directory (see “Analyze Data Tab” on page 4-38 for more information about this option).

If the automatic data export function is NOT enabled and you want to export the data file, use the following procedure.

To export the metrics from an experiment run:

Step Action1 From the File menu in the main window, select Export Metrics.

The Export Metrics dialog box opens.

2 Choose the desired options (see “Configure Metrics Export Dialog Box” on page 4-23 for information on the options in this dialog box).



3 Click OK. The Save As dialog box opens.

In the Save in field, navigate to the directory/folder for which you want the file saved and type a name for the file in the File Name field.

4 Click Save to save the metrics data file.

To export the metrics from an experiment run: (continued)

Step Action


Importing a Metrics File into Microsoft Excel®

Importing a Metrics File into Microsoft Excel®

About ImportingMetrics

Metric data from multiple wells of the PatchPlate or compound plate may be imported into a spreadsheet application such as Microsoft Excel. Opening the data file within Microsoft Excel, will launch the File Import Wizard which will guide you through the process.

Importing MetricData Files

To import metric-data files into Microsoft Excel:

Step Action

1 Open Microsoft Excel application and select Open from the File menu.

2 In the Open File dialog box, navigate to the data file location.

Note Make sure that All Files (*,*) in the Files of Type field is selected

3 Select the metric file that you wish to open (it will have a *.iha file extension) and click Open. This will launch the three-step Import File Wizard.

4 Accept the default settings by clicking Next and Finish.



5 In the example below, the PatchPlate metric-data was selected for import. The second column on the left identifies the PatchPlate well and each subsequent column displays a metric result for the well indicated. Depending on the options chosen for the export, the results may include the native resistance and offset metrics in addition to the user-defined metrics. See the key at the bottom left of the table for an explanation of the letter codes that appear in the results columns.

To import metric-data files into Microsoft Excel: (continued)

Step Action


Shutting Down the System After Completing All Daily Experiments

Shutting Down the System After Completing All Daily Experiments

About ShuttingDown

When you are finished running all experiments for the day, the system should be cleaned and powered down.

IMPORTANT Cleaning the system should be done every day to avoid contamination problems.

Cleaning theSystem

To prepare the system for shut-down

Step Action

1 Wait for all instrument operations to complete and for the traffic light icon on the main window toolbar to turn green


3 Remove all removable reservoirs and compound plates (i.e., Soak, Plate 1, Cells, Buffer, and Plate 2).

4 Discard the contents and clean all removable reservoirs.

5 Install clean and dry v-bottom reservoir into the Buffer station on the experiment stage.

CAUTION Check to verify that the v-bottom reservoir is properly seated in the appropriate station on the experiment stage. Partial seating of the reservoir will damage the electronics and/or fluidics head.

6 Dispense at least 5 mL of 50/50 ethanol/filtered water wash solution into the buffer reservoir.

7 Drain all source bottles.

IMPORTANT Turn the bottle to unscrew the bottle cap when connecting or removing the bottles. All bottle caps are connected to internal tubing lines which will kink if bottle caps are turned.

8 Rinse all source bottles thoroughly with clean, filtered water.

9 Fill all source bottles with 50/50 mixture of ethanol and filtered water. Fill each bottle ~1 inch.

10 Load all source bottles.

IMPORTANT Check that the labeling on the bottle cap matches the labeling on the appropriate source bottle.The bottle cap contains a tubing line that connects to the appropriate station. The bottle cap is distinguished by a label that corresponds to its matching bottle. Installing the incorrect bottle cap to the source bottle will result in flow of incorrect solutions to various stations.

IMPORTANT Turn the bottles when connecting or removing the bottles form the bottle caps. Turning the bottle caps will result in linked-tubing lines.

IMPORTANT Check that all bottles are seated properly in the holding rack positions. All bottles contain a flange at the lower part of the bottle neck. This flange supports the weight of the bottle as it is inserted into the holding rack. Improper seating will damage the bottles and connected tubing.



11 Load an old PatchPlate into the PatchPlate station on the experiment stage:

a. Check and if necessary, dry any traces of fluid on the top surface of the PatchPlate plenum.

CAUTION Keep the top surface of the plenum dry at all times. The top surface of the plenum houses the vacuum port and introducing moisture will damage vacuum pump.

b. Place the PatchPlate into the plenum cavity.

c. Seat the clamping frame on the PatchPlate station.

d. Turn the PatchPlate vacuum switch from the Release to the Hold position.

e. Apply an even force to the clamping frame to create a vacuum seal.

Note The vacuum seal is complete when vacuum air leakage is no longer audible at the clamping interface.


13 Wait for the traffic-light icon on the main window toolbar to turn green.

14 From the Utilities menu, select flush and rinse. The system will perform a flush and rinse cycle to clean the fluidics lines and the 12-channel pipettor.

15 After completion of the flush and rinse cycles, open the instrument access door and remove the v-bottom reservoir from the Buffer station on the experiment stage.

16 Discard the contents and clean the reservoir.

17 Perform the shut-down procedure (refer to “Procedure for Shutting-Down” on page 3-3.)

To prepare the system for shut-down (continued)

Step Action


A
Maintenance A
Appendix Contents

In This Appendix This appendix provides the information and procedures required for maintaining the IonWorks™ HT system. in proper working condition.

Topic See Page

Daily Maintenance A-2

Chloriding the E-Head Probes A-4

Detecting a Salt Bridge A-6

Test Pipettor Utility A-7

IonWorks HT Users Guide A-1

Appendix A

Daily Maintenance

About DailyMaintenance

The procedures below are quick-start procedures that should be followed when using the IonWorks HT system. More detailed operating procedures are found in Chapter 4 and Chapter 5.

Start of Day

Prior to Each Run

End of the Day

Daily maintenance tasks to perform at the start of the day:

Step Action

1 Install previously used PatchPlate.

2 Rinse all bottles with deionized water to remove ethanol residue.

3 Fill all bottles with the appropriate fluid:

a. External = PBS

b. Internal = User choice based upon cells

c. Wash = PBS

d. Antibiotic = Internal solutiona

e. Buffer reservoir = PBS

a. Antibiotic is used in the antibiotic bottle during assay runs.For flushing and rinsing, internal solution is used in the antibiotic bottle.

4 Run Flush and Rinse command.

5 Verify proper vacuum pressure (low and hi vac).

Hi = >23’’ on dial gauge

Lo = 9.3 during most of assay (4.3 during Hole Test).

Note Typical source of leaks are vacuum waste bottles and plenum clamp.

Tasks to perform prior to each run:

Step Action

1 Verify quantity of fluid in each source bottle.

2 Verify that the correct reservoirs are in their location.

3 Verify that the drug plate is seated properly.

4 Remove all covers

5 Verify that all the reservoirs are seated properly.

Tasks to perform at the end of the day:

Step Action

1 Empty all bottles of fluid.

2 Fill (1–2 in.) all bottles with 50% ethanol solution.

3 Fill buffer reservoir with 5 mL of 50% ethanol solution.

4 Run the Flush and Rinse command.

5 Dry out area under PatchPlate (do NOT touch ground electrode).

6 Place used PatchPlate in plenum.

A-2 IonWorks HT Users Guide

Daily Maintenance

PatchPlateContamination

To minimize PatchPlate contamination:

♦ Load PatchPlate just prior to experiment

♦ Clean cell and buffer reservoirs prior to experiment

– Wash with deionized water

– Blow out with clean, dry compressed air.


Appendix A

Chloriding the E-Head Probes‘

Purpose ofChloride

Application

As part of routine maintenance, the 48 silver-plated stainless-steel probes of the E-head must be coated periodically with a chloride layer. Overtime, the repetitive electrical recordings will deplete the E-head probes if this chloride coating and will cause an inevitable degradation in the performance of the E-head.

Applying aChloride Coating

to the E-Head

To apply a chloride coating to the E-head:

Step Action

1 Perform the system start-up procedure (see Chapter 3, “Starting Up and Shutting Down the System.’)


3 Remove and clean the flat-bottom reservoir in the Soak station.

4 Install the flat-bottom reservoir into the Soak position on the experiment stage.

CAUTION Do not install the v-bottom removable reservoirs into the Soak station on the experiment stage. The v-bottom removable reservoirs are allocated for use either in the Cells or the Buffer stations on the experiment stage. The v-bottom reservoirs are designed for accommodating the 12-channel pipettor. The v-bottom reservoirs do not accommodate the 48-channel E-head. Placement of a v-bottom reservoir in the Soak station will damage the E-head.

CAUTION Check and verify that the removable reservoirs are seated properly in the appropriate stations on the experiment stage prior to the start of the maintenance procedure. Partial seating will damage the electronics and/o fluidics head.

5 Fill the Soak reservoir with 10 mL of chloride solution i.e., bleach.

IMPORTANT Do not fill the flat-bottom reservoir in the Soak station with less than 9.8 mL or more than 10 mL of solution. Under-filling can cause inadequate soaking height of the probes on the E-head. Over-filling can immerse and wet the clamping bar which holds the E-head probes and cause electrical short circuit between the probes and E-head.


7 Wait for the traffic-light button on the main window toolbar to turn green and then select Soak probes from the Utilities menu.

8 Type in 60 (sec) for the soaking duration and click OK to start soaking the probes.

The E-head will move to the soak station and immerse the 48 probes into the reservoir for the specified duration. After soaking is completed, the E-head will return to its home position.

9 Wait for the traffic-light button on the main window toolbar to turn green and then open the instrument access door and remove the flat-bottom reservoir from the Soak station.

10 Discard the contents, clean the reservoir and reinstall a clean, dry flat-bottom reservoir into the Soak position on the experiment stage.

CAUTION Do not install a v-bottom reservoir into the Soak station. V-bottom reservoirs do not accommodate the 48-probe E-head and their use will cause damage to the E-head.

CAUTION Check and verify that the reservoir is properly seated in the appropriate station on the experiment stage. Partial seating will damage the E-head.


Chloriding the E-Head Probes

11 Fill the flat-bottom Soak reservoir with 10 mL of external solution (PBS is recommended).

IMPORTANT Do not fill the flat-bottom reservoir in the Soak station with less than 9.8 mL or more than 10 mL of solution. Under-filling can cause inadequate soaking height of the probes on the E-head. Over-filling can immerse and wet the clamping bar which holds the E-head probes and cause electrical short circuit between the probes and E-head.

12 Close the instrument access door.

13 Wait for the traffic-light button on the main window toolbar to turn green and then select Soak from the Utilities menu.

14 Type in 300 (sec) for the soaking duration and then click OK to start the soaking operation.

The E-head will move to the Soak station and immerse the 48 probes into the flat-bottom soak reservoir for the specified duration. After soaking is completed, the E-head will return to its home position.

15 Wait for the traffic-light button on the main window toolbar to turn green, open the instrument access door and remove the flat-bottom reservoir from the Soak station.

16 Discard the contents and if the system will not be used, perform the shut down procedure (see page 3-3 for instructions).

To apply a chloride coating to the E-head: (continued)

Step Action


Appendix A

Detecting a Salt Bridge

Purpose ofSalt-Bridge

Detection

Sometimes salts can accumulate on the electrodes, forming a bridge and causing a short across the pins on the E-Head. This test is designed to detect this problem.

Detecting a SaltBridge

To detect a salt bridge:

Step Action

1 Launch the IonWorks software.

2 Fill the soak boat with 10 mL of PBS.

3 Select Soak Electrodes from the Utilities menu.

4 Enter 300 seconds, when prompted.

5 Upon completion of the utility execution, select Test Electrodes.

6 Click Acquire.

7 If there is no salt bridge, all pins should read N/A. Any other value indicates a short across the pins.


Test Pipettor Utility

Test Pipettor Utility

Purpose of TestPipettor Utility

The Test Pipettor utility allows you to test the dispensing accuracy of the pipettor. A 96-well plate containing an indicator dye (such as tartrazine or fluorescein) is pipetted into a 384-well test plate. The test plate is read and analyzed using a standard absorbance or fluorescence plate reader.

Using the TestPipettor Utility

How to use the test pipettor utility

Step Action

1 Prepare a 0.8 mM tartrazine solution in 100 mL DI water (30 mL required per run). Pipette 300 mL per well into a 96-well source plate. Place into the Plate 1 position in the IonWorks HT system.

2 Prime a clear bottom 384-well test plate with 40 µL of DI Water. Pre-read the test plate in a standard absorbance reader at 426 nm. Place the test plate into the Plate 2 position in the IonWorks HT system.

3 Select Test Pipettor from the Utilities menu and set the desired test parameters in the Pipettor Validation dialog box.

4 After the test is completed, the test plate should be covered, shaken for approx. 12 min, and left to rest for 12 min to allow for complete mixing of the dye.

5 Read the test plate in a standard absorbance plate reader at 426 nm.

6 Detailed instructions, SoftMax Pro (serial number 8700-0001, -0002) and Excel (8700-0003) analysis templates are available on the IonWorks HT v1.5 software CD.


B
Product Warranty Statement A
IonWorks HT Product Warranty Statement

The warranties provided in this statement extend only to the original purchaser or licensee (the “Purchaser”) of an instrument, option (e.g., tip washer, stacker assembly), accessory (e.g., filter), or software (each, a “Product”) purchased or licensed from Molecular Devices Corporation (“MDC”) and installed in the United States by an authorized MDC service representative (“MDC representative”).

InstrumentWarranty

MDC warrants that the instrument purchased from MDC (the “Instrument”) will be free from defects in materials and workmanship for a period of twelve (12) months from the date of installation (as certified by an authorized MDC service representative) or thirteen (13) months from the date of shipment, whichever is shorter, provided that the Instrument has been operated at all times in accordance with the instruction manual and user guide by or under the direct supervision of a certified operator who has attended MDC’s training course for the Instrument.

AccessoryWarranty

MDC warrants that the option, accessory or the media on which a copy of the software is placed provided by MDC (each as “Accessory”) will be free from any defects in materials or workmanship. If an Accessory is acquired at the time the Instrument is purchased, then the warranty period for the Accessory will be the same as the warranty period for the Instrument.

If an Accessory that requires installation is acquired after the Instrument is purchased, but during the warranty period of the Instrument, then the warranty period for the Accessory will be greater of (i) the remaining warranty period for the Instrument, or (ii) the lesser of ninety (90) days from the date of installation of the Accessory or one hundred and twenty (120) days from the date of shipment of the Accessory. If an Accessory that requires installation is acquired after the warranty period for the Instrument has expired, the warranty period for the Accessory will be the lesser of ninety (90) days from the date of installation of the Accessory or one hundred and twenty (120) days from the date of shipment of the Accessory.

If an Accessory that does not require installation is acquired after the Instrument is purchased, but during the warranty period of the Instrument, then the warranty period for the Accessory will be the greater of (i) the remaining warranty period for the Instrument, or (ii) ninety (90) days from the date of shipment of the Accessory. If an Accessory that does not required installation is acquired after the warranty period for the Instrument has expired, the warranty period for the Accessory will be ninety (90) days from the date of shipment of the Accessory.

IonWorks HT Users Guide IonWorks HT Product Warranty Statement-1

Appendix B

WarrantyExclusion

The warranties provided in this Statement will not apply to any Product if MDC determines that a problem (i) arises from or is related to any normal wear and tear, (ii) is caused by accident, abuse, misuse, negligence, misapplication, fire, earthquake, flood, other force major event, failure of electrical power, the use of unauthorized parts or reagents, or unauthorized repairs or modifications, or (iii) is caused during or as a result of shipment or relocation. The warranties provided in this Statement will not apply to any Product if the MDC serial number has been removed or defaced from such Product or to any consumables such as reagent kits and tips.

Remedy For any breach of the warranties provided in this Statement, MDC will, at its own expense and option, and as its sole obligation, and as the Purchaser’s exclusive remedy, (a) repair or replace any defective Product if the Purchaser notifies MDC during the applicable warranty period and an MDC representative determines that the Product is defective and is covered by the warranty; or (b) if MDC determines that it is unable to repair or replace the defective Product, refund the purchase price paid by the Purchaser for the defective Product. The warranty period for the repaired or replaces Product will not exceed the warranty period the defective Product. The warranty period for any replacements parts, which may be new or reconditioned at MDC’s sole discretion, will not exceed the warranty period for the defective Product.

WarrantyDisclaimer

THE FOREGOING WARRANTIES ARE EXCLUSIVE AND IN LIEU OF ALL OTHER WARRANTIES, EITHER EXPRESS, IMPLIED, OR STATUTORY, REGARDING THE PRODUCTS, INCLUDING ANY WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, TITLE, AND NON-INFRINGEMENT OF THIRD PARTY RIGHTS.

IonWorks HT Product Warranty Statement-2 IonWorks HT Users Guide

C
Customer Support A
Contacting Molecular Devices

Contacting via theWeb

For customer support information and worldwide distributors information, go to MDC’s web at www.moleculardevices.com

Contacting byTelephone, Fax or

Email

For Molecular Devices Corporation main office, use the following contact information:

Telephone

408-747-1700 or 800-747-5577

Main Fax

408-747-3601

Customer Orders Fax

408-747-3558

Technical Support Fax

408-747-3603

E-mail

[email protected]

IonWorks HT Users Guide Contacting Molecular Devices-1

D
Mapping Diagrams A
Fluidics Head Mapping Diagram

Table D-1 Numbers in the table show dispense order for buffer and cell additions. F-Head needle (NDL) position is indicated in top row. PatchPlate row and column (R/C) are also indicated. Underlined "1" indicates the first of 32 replicate dispenses by the fluidics head.

F Head NDL 1 2 3 4 5 6 7 8 9 10 11 12

PatchPlateR/C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

A 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

B 5 6 7 8 5 6 7 8 5 6 7 8 5 6 7 8 5 6 7 8 5 6 7 8 5 6 7 8 5 6 7 8 5 6 7 8 5 6 7 8 5 6 7 8 5 6 7 8

C 9 10 11 12 9 10 11 12 9 10 11 12 9 10 11 12 9 10 11 12 9 10 11 12 9 10 11 12 9 10 11 12 9 10 11 12 9 10 11 12 9 10 11 12 9 10 11 12

D 13 14 15 16 13 14 15 16 13 14 15 16 13 14 15 16 13 14 15 16 13 14 15 16 13 14 15 16 13 14 15 16 13 14 15 16 13 14 15 16 13 14 15 16 13 14 15 16

E 17 18 19 20 17 18 19 20 17 18 19 20 17 18 19 20 17 18 19 20 17 18 19 20 17 18 19 20 17 18 19 20 17 18 19 20 17 18 19 20 17 18 19 20 17 18 19 20

F 21 22 23 24 21 22 23 24 21 22 23 24 21 22 23 24 21 22 23 24 21 22 23 24 21 22 23 24 21 22 23 24 21 22 23 24 21 22 23 24 21 22 23 24 21 22 23 24

G 25 26 27 28 25 26 27 28 25 26 27 28 25 26 27 28 25 26 27 28 25 26 27 28 25 26 27 28 25 26 27 28 25 26 27 28 25 26 27 28 25 26 27 28 25 26 27 28

H 29 30 31 32 29 30 31 32 29 30 31 32 29 30 31 32 29 30 31 32 29 30 31 32 29 30 31 32 29 30 31 32 29 30 31 32 29 30 31 32 29 30 31 32 29 30 31 32

IonWorks HT Users Guide D-1

Electronics Head Mapping diagram
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2 C

16R

2 C

16R

2 C

17R

2 C

17R

2 C

18R

2 C

18R

2 C

19R

2 C

19R

2 C

20R

2 C

20R

2 C

21R

2 C

21R

2 C

22R

2 C

22R

2 C

23R

2 C

23R

2 C

24R

2 C

24


Appendix D

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1112

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1516

1718

1920

2122

2324

2526

2728

2930

3132

3334

3536

3738

3940

4142

4344

4546

4748

AA

1 #1

B1

#1A

1#2

B1

#2A

2#1

B2

#1A

2#2

B2

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3#1

B3

#1A

3#2

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#2A

4#1

B4

#1A

4#2

B4

#2A

5#1

B5

#1A

5 #2

B5

#2A

6 #1

B6

#1A

6 #2

B6

#2A

7 #1

B7

#1A

7 #2

B7

#2A

8 #1

B8

#1A

8 #2

B8

#2A

9 #1

B9

#1A

9 #2

B9

#2A

10

#1B

10

#1A

10

#2B

10

#2A

11

#1B

11

#1A

11

#2B

11

#2A

12

#1B

12

#1A

12

#2B

12 #2

BC

1 #1

D1

#1C

1#2

D1

#2C

2#1

D2

#1C

2#2

D2

#2C

3#1

D3

#1C

3#2

D3

#2C

4#1

4D #1C

4#2

D4

#2C

5#1

D5

#1C

5 #2

D5

#2C

6 #1

D6

#1C

6 #2

D6

#2C

7 #1

D7

#1C

7 #2

D7

#2C

8 #1

D8

#1C

8 #2

D8

#2C

9 #1

D9

#1C

9 #2

D9

#2C

10 #1D

10 #1C

10 #2D

10 #2C

11 #1D

11 #1C

11 #2D

11 #2C

12 #1D

12 #1C

12 #2D

12 #2

CE

1 #1

F1

#1E

1 #2

F1

#2E

2 #1

F2

#1E

2 #2

F2

#2E

3 #1

F3

#1E

3 #2

F3

#2E

4 #1

F4

#1E

4 #2

F4

#2E

5 #1

F5

#1E

5 #2

F5

#2E

6 #1

F6

#1E

6 #2

F6

#2E

7 #1

F7

#1E

7 #2

F7

#2E

8 #1

F8

#1E

8 #2

F8

#2E

9 #1

F9

#1E

9 #2

F9

#2E

10

#1F

10

#1E

10

#2F

10

#2E

11

#1F

11

#1E

11

#2F

11

#2E

12

#1F

12

#1E

12

#2F

12 #2

DG

1 #1

H1

#1G

1 #2

H1

#2G

2 #1

H2

#1G

2#2

H2

#2G

3 #1

H3

#1G

3#2

H3

#2G

4#1

H4

#1G

4#2

H4

#2G

5#1

H5

#1G

5 #2

H5

#2G

6 #1

H6

#1G

6 #2

H6

#2G

7 #1

H7

#1G

7 #2

H7

#2G

8 #1

H8

#1G

8 #2

H8

#2G

9 #1

H9

#1G

9 #2

H9

#2G

10

#1H

10

#1G

10

#2H

10

#2G

11

#1H

11

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11

#2H

11

#2G

12

#1H

12

#1G

12

#2H

12 #2

EA

1 #3

B1

#3A

1#4

B1

#4A

2#3

B2

#3A

2#4

B2

#4A

3#3

B3

#3A

3#4

B3

#4A

4#3

B4

#3A

4#4

B4

#4A

5#3

B5

#3A

5 #4

B5

#4A

6 #3

B6

#3A

6 #4

B6

#4A

7 #3

B7

#3A

7 #4

B7

#4A

8 #3

B8

#3A

8 #4

B8

#4A

9 #3

B9

#3A

9 #4

B9

#4A

10

#3B

10

#3A

10

#4B

10

#4A

11

#3B

11

#3A

11

#4B

11

#4A

12

#3B

12

#3A

12

#4B

12 #4

FC

1 #3

D1

#3C

1#4

D1

#4C

2#3

D2

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2#4

D2

#4C

3#3

D3

#3C

3#4

D3

#4C

4#3

4D #3C

4#4

D4

#4C

5#3

D5

#3C

5 #4

D5

#4C

6 #3

D6

#3C

6 #4

D6

#4C

7 #3

D7

#3C

7 #4

D7

#4C

8 #3

D8

#3C

8 #4

D8

#4C

9 #3

D9

#3C

9 #4

D9

#4C

10 #3D

10 #3C

10 #4D

10 #4C

11 #3D

11 #3C

11 #4D

11 #4C

12 #3D

12 #3C

12 #4D

12 #4

GE

1 #3

F1

#3E

1 #4

F1

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2 #3

F2

#3E

2 #4

F2

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3 #3

F3

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3 #4

F3

#4E

4 #3

F4

#3E

4 #4

F4

#4E

5 #3

F5

#3E

5 #4

F5

#4E

6 #3

F6

#3E

6 #4

F6

#4E

7 #3

F7

#3E

7 #4

F7

#4E

8 #3

F8

#3E

8 #4

F8

#4E

9 #3

F9

#3E

9 #4

F9

#4E

10

#3F

10

#3E

10

#4F

10

#4E

11

#3F

11

#3E

11

#4F

11

#4E

12

#3F

12

#3E

12

#4F

12 #4

HG

1 #3

H1

#3G

1 #4

H1

#4G

2 #3

H2

#3G

2#4

H2

#4G

3 #3

H3

#3G

3#4

H3

#4G

4#3

H4

#3G

4#4

H4

#4G

5#3

H5

#3G

5 #4

H5

#4G

6 #3

H6

#3G

6 #4

H6

#4G

7 #3

H7

#3G

7 #4

H7

#4G

8 #3

H8

#3G

8 #4

H8

#4G

9 #3

H9

#3G

9 #4

H9

#4G

10

#3H

10

#3G

10

#4H

10

#4G

11

#3H

11

#3G

11

#4H

11

#4G

12

#3H

12

#3G

12

#4H

12 #4


Index
A
active protocol dialog box, description 4-46analysis

metrics summary, viewing 4-18metrics, designing 1-3results, viewing 1-3

analyze data tab, description 4-38

Cchemical

safety 1-6waste hazard 1-6

components overview of the system 1-3compound plate

main window view 4-3main window viewing 4-16well data, viewing 4-17

configureanalysis method pane, description 4-13filter metrics pane, description 4-12hits display, description 4-14metrics export dialog box, description 4-23

Ddata

exporting 5-23viewing 1-3viewing from an experiment 5-16

data file title bar, description 4-9data menu, description 4-8design analysis metric dialog box, description 4-39dialog boxes

access to end 4-28analyze data 4-38configure metrics export 4-23design analysis metric 4-39edit metrics 4-42experiment summary 4-47experimental protocol properties 4-24 to 4-42file menu 4-20 to 4-23name file 4-24open data 4-20save as 4-21set single addition screen parameters 4-34start to seal test 4-25waveform editor 4-31

dialog boxes and windowsabout 4-19trace groups 4-48view menu 4-46 to 4-50

Eedit menu, description 4-6

edit metrics menu dialog box, description 4-42electrical measurements, designing 1-3experiment menu, description 4-7experiment protocol properties

name file tab 4-24experiment summary dialog box, description 4-47experimental protocol properties

access to end tab 4-28start to seal test 4-25

experimental protocol properties dialog boxes 4-24 to 4-42exporting data 5-23

Ffile menu dialog boxes 4-20 to 4-23file menu, description 4-5

Hhelp menu, description 4-9holding potential option 4-33holding time option 4-33how the IonWorks HT system works 1-2

Iintroduction

safety 1-6 to 1-7safety alert symbols 1-8

Mmain window

configure analysis method pane description 4-13configure filter metrics pane, description 4-12configure hits display description 4-14PatchPlate description 4-10plate legend and statistics area description, plate

legend and statistics area, description 4-11toolbar description 4-9view compound plate 4-3view PatchPlate 4-2viewing analysis metrics summary 4-18viewing compound plate 4-16viewing compound-plate well data 4-17

main window menudata 4-8edit 4-6experiment 4-7file 4-5help 4-9utilities 4-7window 4-8

menu bar, description 4-5MSDSs

information 1-6ordering 1-7

IonWorks HT User Guide 1

Index

Oopen data dialog box, description 4-20operating the IonWorks HT system, overview 1-3operating the system

exporting the data 5-23setting up a protocol 5-12setting up the instrument for an experiment 5-3 to

5-15setting up the software and starting the

experiment 5-12 to 5-18shutting down after an experiment 5-27viewing the data from an experiment 5-16

overviewdesigning analysis metrics 1-3designing electrical measurement parameters 1-3IonWorks HT system components 1-3operating the IonWorks HT system 1-3PatchPlate usage 1-3viewing analysis results 1-3viewing data 1-3

overview of the IonWorks HT system 1-2, 3-2

PPatchPlate

area description 4-10main window view 4-2usage, overview 1-3

pipettor options 4-36, 4-37post-signal holding time option 4-33protocol, setting up 5-12

Rrequirements of the system 1-5running an experiment

exporting the data 5-23setting up a protocol 5-12setting up the instrument 5-3 to 5-15shutting down the system 5-27

running the experimentsetting up the software and starting 5-12 to 5-18

Ssafety 1-6 to 1-8

alert symbols 1-8chemical hazard 1-6chemical waste hazard 1-6documentation user words 1-6instrument labels 1-7MSDSs, about 1-6MSDSs, ordering 1-7operating precautions 1-7warnings 1-6

salt bridge, detecting A-6save as dialog box 4-21set single addition screen parameters dialog box 4-34setting up the instrument for an experiment 5-3 to 5-15setting up the system

exporting the data 5-23setting up a protocol 5-12setting up the software and starting the

experiment 5-12 to 5-18viewing the data 5-16

short, from a salt bridge A-6shutting down the system 5-27solvent wash 4-36, 4-37specifications and requirements of the system 1-5status bar, description 4-4system components 1-3system requirements and specifications 1-5

Ttitle bar, description 4-4toolbar, main window description 4-9trace groups window 4-48

Uutilities menu, description 4-7

Vview active protocol dialog box, dialog boxes

view active protocol 4-46view menu dialog boxes and windows 4-46 to 4-50view menu, description 4-6viewing data, overview 1-3

Wwash, after compound addition 4-36, 4-37waveform editor dialog box, description 4-31window menu, description 4-8windows and dialog boxes, about 4-19working principles of the IonWorks HT system 1-2

2 IonWorks HT User Guide

Molecular Devices Corporation.1311 Orleans DriveSunnyvale, CA 94089Tel: 409-7471700www.moldev.com

ionworks ht - mdc.custhelp.com

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