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Thermo Scientific ToxInsight Consumer and Health

Products Safety Program

Endocrine Profiler Panel

Assay Guide

Thermo Scientific ToxInsight

Endocrine Profiler Panel (EPP)

Assay Guide

Estrogen Receptor alpha and Androgen Receptor Assays

Version 1.0

PUBLISHED BY

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P/N LC07213500

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Table of Contents

Chapter 1 Introduction ............................................................................................................. 1

Terminology, Conventions, and Symbols .............................................................................. 1

Terminology ................................................................................................................... 1

Conventions ................................................................................................................... 2

Symbols ......................................................................................................................... 2

Contacting Technical Support ............................................................................................... 2

Documentation Feedback ..................................................................................................... 3

Chapter 2 Overview .................................................................................................................. 5

The ERα Redistribution Assay .............................................................................................. 6

The AR Redistribution Assay ................................................................................................ 7

References ........................................................................................................................... 9

Chapter 3 Endocrine Profiler Panel Assay Principle ............................................................ 11

EPP Assay Workflow .......................................................................................................... 11

Treating, Labeling, and Quantitatively Imaging Cells ..................................................... 12

Using Template Analysis to Make Predictions for Potential Endocrine Disruption .......... 16

Using Quantitatively Imaged Data to Make the Proper Dose Range Prediction .............. 17

Using Quantitatively Imaged Data to Evaluate Receptor Activity ................................... 21

Stepwise Extrapolation of Response Predictions for both Templates ............................. 23

Chapter 4 Cell Imaging .......................................................................................................... 39

Fixed Exposure versus Autoexposure ........................................................................... 39

Ensuring that Wells without Cells are not Rejected........................................................ 40

Object Selection and Gating ......................................................................................... 40

Chapter 5 Post-Imaging Analysis for EPP Prediction .......................................................... 41

Microsoft Excel Template for the EPP Assay ....................................................................... 41

Guidelines .......................................................................................................................... 41

Installing the Template ........................................................................................................ 42

Template Workflow ............................................................................................................. 43

Loading the Template ......................................................................................................... 43

Configuring the Data Source ............................................................................................... 45

Configuring the Data Source for Store Database Retrieval ............................................ 46

Configuring the Data Source for Local Plate Data Retrieval ........................................... 47

Selecting Plates .................................................................................................................. 48

Entering Experimental Design Information........................................................................... 49

II

Running the Template ........................................................................................................ 52

Worksheets ........................................................................................................................ 53

Worksheet 1: Rawdata ................................................................................................. 53

Worksheet 2: Basal Response ...................................................................................... 54

Worksheet 3: Normalized Data ..................................................................................... 57

Worksheet 4: Normalized Response ............................................................................. 58

Worksheet 5: QC .......................................................................................................... 59

Worksheet 6: Graphs ................................................................................................... 60

Worksheet 7: Conclusion .............................................................................................. 61

Suggested User Template Worksheet Workflow ........................................................... 63

Chapter 6 Sample Preparation Using the EPP ERα and AR Assays ................................... 65

Important Information.......................................................................................................... 65

1

Introduction

The Thermo Scientific ToxInsight IVT Platform offers a predictive, cell based imaging

approach to determining the toxicity risk of a compound through the measurement of multiple biomarkers in individual cells. The automated, bench-top, easy to use platform allows a

researcher to benefit from physiologically relevant assays early in the drug discovery process,

rather than traditional late stage histopathology or animal studies.

The ToxInsight™ Endocrine Profiler Panel (EPP) Assays, through the use of the ToxInsight

Estrogen Receptor alpha (ER ) and Androgen Receptor (AR) Assay Cartridges, identify potential compound-induced endocrine disruption by simultaneous detection and analysis of

multiplexed cellular targets and properties. These are cell loss, general nuclear intensity and

morphological features along with nuclear receptor foci formation and potential degradation or translocation of proteins. The EPP Assays deliver robust, convenient, reproducible results, and

can be used for routine assessments of potential estrogenic or androgenic compounds.

This guide describes the principles of the EPP Assay, sample preparation, cell imaging, and post-imaging analysis for prediction of potential endocrine disruptors.

Terminology, Conventions, and Symbols

This guide assumes basic user knowledge of computers using the Microsoft® Windows®

operating system and experience working with windows, menus, commands, buttons, tabs,

dialog boxes, and other Microsoft Windows elements. If you are unfamiliar with these terms,

please refer to Microsoft Windows documentation.

Throughout this guide certain terminology, conventions, and symbols are used consistently.

These conventions are described in the following sections.

Terminology

Term Definition

Click This term means to place the mouse pointer over the item, then depress and release the primary

mouse button (usually the left button) in one quick motion

Right-click This term means to place the mouse pointer over the item, then depress and release the secondary mouse button (usually the right button) in one quick motion

Double-click This term means to place the mouse pointer over the item, then depress and release the primary

mouse button twice in quick succession.

Drag This term means to place the mouse pointer over the item, depress and hold down the left mouse

button, move the pointer (and the object) to some target location, then release the mouse button.

Press This term means to push and release a key on the keyboard. For example, press the Tab key.

Shift Ctrl Alt When any of these terms appear before any of the above terms, it means to hold down the

specified keyboard key while taking the hyphenated action. Thus, Shift-click means to hold down the Shift key while clicking an item.

Shortcut menu This is a menu that appears when you right-click an item.

Chapter

1

2 Chapter 1 Introduction

Conventions

Menu names, menu items, buttons, and options appear in bold type. For example: The location

of the files is C:\CellData.

Window titles and dialog box names begin with uppercase letters.

Symbols

The following symbol appears throughout this document in order to draw your attention to

important information such as operating tips and suggestions.

Symbol Description

A tip, suggestion, or additional information.

Contacting Technical Support

If you have a technical question that you are unable to answer after consulting the

documentation, please contact Technical Support.

Thermo Fisher Scientific

100 Technology Drive

Pittsburgh, Pennsylvania 15219

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Before contacting Technical Support for service or support, it is helpful if you are prepared to

answer the following questions:

What were you doing when the problem occurred?

Can you reproduce the problem?

Did you try to solve the problem? If so, what steps did you take and what did you

observe?

Which error messages, if any, appeared?

Having these answers will help us provide you with a solution as quickly as possible.

Chapter 1 Introduction 3

Documentation Feedback

Thermo Fisher Scientific is committed to providing you with the highest quality product

documentation and online help and we welcome comments and suggestions for improvement.

Before emailing your feedback to us, it is helpful if you include the following information:

For printed manuals, provide the document title and part number. The part number can be

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5

Overview

Endocrine disruptors are compounds that alter steroid activity thereby perturbing endocrine

system functionality. Environmental endocrine disruptors have been linked to numerous adverse health effects and reproductive problems in both humans and wildlife [1, 2]. The

advent of complex compound libraries in addition to a large catalog of existing compounds

necessitates an automated procedure to assess a compound‘s in vitro endocrine activity prior to investigating potential organismal and environmental impact. Utilization of in vitro screens for

potential endocrine active compounds (EACs) can reduce animal testing by categorizing and

prioritizing compounds based on their ability to alter endocrine receptor activity.

Bioethics, efficiency, and cost of animal testing have been moving the trend towards in vitro cell-based assays to replace protocols using animal tissues, to reduce animal usage by

predicting potential in vivo action, and to provide additional toxicity information. The need for

more sensitive assays that can provide insight into mechanisms of action for endocrine receptor activation extends to environmental and industrial safety as well as consumer products

from cosmetics to food additives. A goal of in vitro testing is to detect compounds with

potential endocrine activity and assess the magnitude and potency of the response. Because of diverse action mechanisms with different compounds, monitoring multiple, independent

outputs in the same cell increases the predictive power of the assay.

Thermo Fisher Scientific provides the Thermo Scientific ToxInsight IVT Platform as a

complete solution for cell-based predictive toxicology. The ToxInsight IVT Platform complete solution comprises of a cellular imaging instrument, software, reagents and other

consumables. For this platform to offer ease-of-use performance, the biology and

instrumentation are linked by the ToxInsight EPP Assay Cartridges, an integral part of the ToxInsight IVT Platform. The ToxInsight Cartridges are reagent kits relevant to endocrine

receptor activity and associated software assay protocols that enable the specific kit to be run

on the ToxInsight instrument.

The ToxInsight IVT Platform offers a predictive, cell-based imaging approach to determining

the potential endocrine disruption risk of a compound through the measurement of multiple

biomarkers in individual cells. The automated, bench-top, easy to use platform allows a

researcher to benefit from physiologically relevant assays early in the screening process, rather than in late stage animal studies. Because of diverse action mechanisms with different

compounds, directly monitoring receptor activation in the same cell increases the assay‘s

predictive power. The data acquired using the ToxInsight EPP Assay Cartridge should be analyzed with the provided Thermo Scientific AddIn for Microsoft

® Excel

® and Excel

Template for the EPP Assay to predict endocrine disruption from the tested drugs or

compounds.

The ToxInsight EPP Assay Cartridge is used for identifying compound effects to endocrine disruption by simultaneous detection of multiplexed targets (by using the DNA binding dye,

Hoechst 33342) including cell loss, nuclear DNA, morphological characteristics, as well as

nuclear GFP foci formation, along with potential degradation (for ER ) or translocation of

Chapter

2

6 Chapter 2 Overview

receptor (AR) using an end-point assay based on Thermo Scientific Redistribution technology

and fluorescence detection in fixed cells grown on standard high-density microplates.

The ToxInsight EPP ER and AR Assay Cartridges consist of the following:

Optimized reagents which include the Thermo Scientific CryoRedi cells, media,

reagents, plates, and fluorescent probes for the monitored cell targets

Respective ToxInsight EPP Assay Analysis Templates, which are Microsoft Excel®

templates, that analyze and interpret the imaging data to arrive at general toxicity and

dose range predictions as well as receptor activity and potential mimic characteristics

of each evaluated sample compound

The ToxInsight EPP Assay uses both ER and AR Assay Cartridges with two separate Excel-based templates each. The first template type identifies possible toxic concentrations of each compound through use of general viability features (cell count, nuclear size, and nuclear

intensity) with respect to receptor response. This template helps you decide what concentration

to use for detailed evaluation of compound-induced receptor activity. The second template provides a detailed analysis of the receptor response and mechanism of action for each

compound by comparison of their dose response to positive controls with respect to vehicle

controls. Here, you can compare the potency and magnitude of the selected compound‘s

primary response (nuclear foci formation) via thresholds of the positive and weak response control, as well as comparing foci formation to an additional receptor response (receptor

degradation for ER and translocation for AR) using the mimic function.

The ToxInsight Endocrine Profiling Panels deliver robust, convenient, reproducible results,

and can be used for routine estrogenic and/or androgenic assessment of compounds. The ER and AR Assay Cartridges include Redistribution™ CryoRedi™ cells, along with media,

reagents, assay protocol, and analysis and data reporting capabilities to predictively profile compounds for potential endocrine disruption. The Redistribution technology monitors the

cellular translocation of GFP-tagged proteins in response to compounds or other stimuli and

allows easy acquisition of multiple readouts from the same cell in a single assay run in addition

to supplementary information about cell morphology, compound fluorescence, and cellular toxicity. CryoRedi cell lines (based on Redistribution technology) are ―ready-to-use‖, where

frozen cells are thawed, plated immediately, and used directly in high content analysis

applications, including profiling and screening studies.

The ERα Redistribution Assay

Estrogen is a regulator of normal endocrine function. Signal transduction induced by estrogens

such as estradiol, the main endogenous human estrogen, is mediated by the estrogen receptor

(ER). ER is a nuclear receptor that upon ligand binding organizes into homo- and heterodimers of the ERα and ERβ subtypes. Following ligand binding, ER acts as a transcription factor and

regulates expression of several target genes such as cyclin D1 and IGF-1 [3]. Estrogens and

estrogen receptors are implicated in development and progression of breast cancer. Moreover, environmental chemical contaminants with estrogenic activity are suggested to promote

reproductive disorders. The ERα Redistribution assay monitors accumulation of an EGFP-

ERα fusion protein in nuclear foci as a measure of ERα activation (Figure 1 and 3A). The

agonist version of the assay uses the potent estrogen 17β-estradiol as a reference compound. The EC50 of 17β-estradiol for nuclear foci formation in the assay is approximately 0.3 nM and

corresponds well with EC50 values for receptor activation reported by others [4].

Chapter 2 Overview 7

Figure 1. Illustration of ERα Redistribution upon stimulation with 17β-estradiol

The AR Redistribution Assay

The androgen receptor (AR) is a nuclear receptor transcription factor that mediates the cellular

actions of androgens, the male sex steroids testosterone and dihydrotestosterone (DHT).

Androgen signaling mediates male sexual differentiation, sexual maturation, and

spermatogenesis. Androgens maintain normal prostate homeostasis but are also involved in

prostate tumorigenesis [5]. Androgens exert their activity through binding to AR resulting in

either activation or repression of androgen responsive genes. Ligand binding exposes a nuclear

localization signal (NLS) allowing translocation of AR to the nucleus via a transport protein. Agonists as well as most antagonists induce translocation of AR to the nucleus. However, only

agonists activate AR functions by interaction with androgen response elements (AREs).

Moreover, AR agonists often induce nuclear localization of AR with a punctuate distribution pattern, the so-called nuclear foci [5, 6, 7]. The AR Redistribution Assay is designed to assay

for compounds inducing EGFP-AR nuclear translocation and formation of nuclear foci. AR

nuclear accumulation and foci formation is promoted by the agonist DHT, which is used as reference compound (Figure 2 and 3B). The EC50 of DHT for nuclear foci formation in the

assay is approximately 0.2 nM.

Figure 2. Illustration of AR Redistribution upon stimulation with DHT

17β-estradiol

Unstimulated cell:

EGFP-ERα localized in the nucleus

Stimulated cell: EGFP-ERα localized

in nuclear foci

Unstimulated cell: EGFP-AR localized both in

cytoplasm and nucleus

Stimulated cell:

EGFP-AR localized in nucleus. Foci formation is evident

DHT

8 Chapter 2 Overview

Figure 3. Inset visualization of GFP foci in ERα (A) and AR (B) cells (20X objective). Cells treated

with β-estradiol (A, bottom) or DHT (B, bottom) show an accumulation of spots within the nuclear region. AR cells also show translocation from the cytoplasm to the nucleus. Images were acquired using the Thermo Scientific ToxInsight IVT Platform. Overlays in the bottom right images represent visualization of the

Compartmental Analysis BioApplication to identify spots (green, A & B) and cytoplasmic area for calculating translocation (orange, B).

A challenge to current in vitro assays used to assess potential endocrine activity is that

compounds may induce toxicity or other non-receptor mediated side effects resulting in data

that does not directly evaluate endocrine activity. Assessing a compound‘s potential for interacting with the endocrine system should incorporate both toxicity and receptor binding

data. The Thermo Scientific EPP Assays address this issue by directly monitoring

fluorescently tagged endocrine receptors and a nuclear stain over a range of compound

concentrations (i.e., dose response) via automated image analysis which allows the

visualization of distinct steps in the receptor activation pathway.

Thus, a major advantage of these assays is the simultaneous monitoring and analysis of

toxicity along with the direct visualization of endocrine receptor activity over a range of

compound concentrations. This provides receptor response data for use in endocrine disruption predictions coupled to toxicity assessment and a secondary receptor read-out to determine the

compound‘s mechanism of receptor activation.

Chapter 2 Overview 9

References

1. Diamanti-Kandarakis, E. et al. 2009. Endocrine Reviews. 30: 293-342.

2. Hotchkiss, A. K. et al. 2008. Toxicological Sciences. 105: 235-259.

3. Levin, E. R. 2005. Molecular Endocrinology. 19(8): 1951-1959.

4. Wilson, S. V. et al. 2004. Toxicological Sciences. 81: 69-77.

5. McEwan, I. J. 2004. Endocrin-Related Cancer. 11: 281-293.

6. Rosenfeld, M. G. and C. K. Glass. 2001. J. Biol. Chem. 276: 36865-36868.

7. Mainwaring, W. I. 1977. Monogr. Endocrinol. 10: 1-178.

8. Association for the Advancement of Medical Instrumentation. Biological evaluation of medical devices – Part 5: Tests for in vitro cytotoxicity. ANSI/AAMI/ISO 10993-5:2009

(2009).

9. Alarid, E.T. 2006. Molecular Endocrinology. 20(9): 1972-1981.

11

Endocrine Profiler Panel Assay Principle

This chapter describes the principles of the ER and AR Assays including a description of the assay workflow, treating, labeling, and imaging cells and using data to make receptor

activation predictions.

EPP Assay Workflow

Both the ER and AR assay methods use their respective CryoRedi cells (U2OS cell lines

expressing either GFP-ER or GFP-AR), Nunc™ Edge 96-well microplates, media and

reagents (Assay Cartridge reagents), an automated, quantitative cell imaging system (ToxInsight IVT Platform), optimized assay and imaging protocols (Compartmental Analysis

supplied on the ToxInsight IVT Platform), and decision-making analysis tools to predict

compound toxicity and receptor activity using the quantitative data from the cell image (Dose Range and Receptor Activity Analysis Templates, which are part of the ToxInsight EPP Assay

Cartridges). The optimized reagents, experimental workflow, and decision-making software

make this a convenient and robust assay to implement and routinely use.

The ToxInsight EPP Assay has the following three key steps (see Figure 4):

1) Sample Preparation: The first step contains an optimized protocol for plating and

compound treatment, followed by fluorescent nuclear labeling of cells with the

optimized reagents from the ToxInsight ER /AR Assay Cartridges. Additional

information is given in Chapter 6.

2) Imaging & Analysis: The second step involves imaging the cells on the ToxInsight

IVT Platform, an automated, quantitative analysis of the imaged cellular targets to

detect changes associated with general toxicity and receptor activity. The samples in

96-well plates are automatically imaged and analyzed by the ToxInsight IVT Platform using the supplied Compartmental Analysis optimized imaging protocols. Additional

information is given in Chapter 4 as well as in the Compartmental Analysis

BioApplication Guide.

3) Dose Range and/Receptor Activity Prediction: The third step applies a set of ToxInsight EPP Templates on the quantitative, multiparametric cell target data

acquired from the previous step. Determination of what concentration (if any) a

compound of interest is toxic with respect to foci formation is accomplished through

the ER and AR Dose Range templates. The Receptor Activity templates compare each compound to appropriate Weak Response and Positive Controls to determine its potency as well as general functionality (potential mimic) against the Positive Control.

Additional information can be found within this chapter as well as in Chapter 5.

Chapter

3

12 Chapter 3 Endocrine Profiler Panel Assay Principle

Dose Range:

Receptor Activity:

Figure 4. Thermo Scientific EPP Assay Steps for Dose Range (top) and Receptor Activity Assays (bottom)

Treating, Labeling, and Quantitatively Imaging Cells

Two separate assays must be performed to ensure that the proper concentration range has been

selected to monitor receptor activity. This is briefly described above in Figure 4.

All of the EPP Assays are performed using CryoRedi cells which have been optimized for immediate plating following thawing from liquid nitrogen and do not need to be cultured prior

to testing. Cells are removed from liquid nitrogen, added to Plating Media, counted and plated

into 96-well microplates. For these assays, the Nunc Edge plates are used to reduce potential

plate edge effects during cell culture and provide low background fluorescence during

imaging.

Automatically acquire images of labeled cells on ToxInsight

IVT Platform & perform automatic

on-the-fly analysis of images

Plate CryoRedi cells onto Nunc 96-well Edge plates (6 plates for 6

compounds) with Plating Media; incubate overnight

Treat cells with DR of selected compounds (using concentration from Dose Range), Weak Response, Max Positive Control, and Positive Controls in Assay Media and incubate 24 h (AR)

or 20 h (ER )

Fix cells and stain with Hoechst using Cartridge reagents

STEP 1: SAMPLE PREPARATION STEP 2: IMAGING & ANALYSIS STEP 3: RECEPTOR ACTIVITY PREDICTION

Import data into appropriate Receptor Activity Analysis Template

Receptor Activity Template will calculate potency of compound

compared to Weak Response and Max Positive Control thresholds as well as if the compound mimics the Positive

Control in foci formation and

degradation (ER )/translocation (AR)

Compare duplicate plates

Automatically acquire images of labeled cells on ToxInsight

IVT Platform & perform automatic

on-the-fly analysis of images

Plate CryoRedi cells onto Nunc 96-well Edge plates (1 plate per 6 compounds) with Plating Media; incubate overnight

Treat cells with selected DR compounds and Positive Controls in Assay Media and incubate 24 h (AR)

or 20 h (ER )

Fix cells and stain with Hoechst using Cartridge reagents

STEP 1: SAMPLE PREPARATION STEP 2: IMAGING & ANALYSIS STEP 3: DOSE RANGE PREDICTION

Import data into appropriate Dose Range Assay Analysis Template

Dose Range Assay Analysis Template will calculate suggested compound concentration for Receptor Activity

Assay based on threshold values for each target vs. vehicle and positive

control responses

Max concentration that fits toxicity and foci formation will be used for setting

up Receptor Activity plates

Chapter 3 Endocrine Profiler Panel Assay Principle 13

Dose Range Assays for both ER and AR

One vial of CryoRedi cells is needed to test six compounds using the Dose Range Assay (seven doses in duplicate at 1:10 dilution + the Maximum Positive Control concentration and a

Vehicle Control). Figure 5 represents the suggested plate layout for this assay.

Figure 5. Suggested plate setup for the Dose Range Assay Plate. Only one plate is needed per six

compounds in duplicate columns. Compounds are added on top of Plating Media to give 1x final dilutions.

For general terminology, the EPP Assay protocol requires that the cells be subjected to one of

the following types of treatment conditions for a specific period of time (24 or 20 hour

compound treatment for AR or ER cells, respectively). For the Dose Range assay, the

following well types can be found:

1) Vehicle Control - VC (e.g., DMSO): used to normalize the data for toxicity predictions

from nuclear measurements and as a background response (lower limit or 0% response)

for GFP receptor measurements.

2) Max Positive Control - MaxPC (i.e., -estradiol for ER or DHT for AR): used to

normalize the data for the GFP receptor responses (upper limit or 100% response).

3) Sample compounds - Sample: potential estrogenic/androgenic compounds that are being

assessed where toxicity and receptor effects are unknown, or are known compounds used

as comparison. Concentrations start at 100 µM (unless limited to a lower starting

concentration due to insolubility) followed by 6 additional 1:10 dilutions.

After treatment, the cells are fixed, washed and stained with Hoechst from the EPP Reagent

Cartridge(s). Images of the fluorescently labeled cells are automatically acquired on the

ToxInsight IVT Platform to detect the different fluorescently labeled cellular targets, and then stored and automatically analyzed on-the-fly with the Thermo Scientific Compartmental

Analysis BioApplication. The Compartmental Analysis BioApplication makes the following

measurements in the cell images (Table 1):

Cell Viability is calculated by measuring the number of nuclei in the images normalized

to the vehicle control (set to 100% viability); a user-defined threshold flags compound

concentrations that decrease nuclei count below the threshold (default set to 80% viability)

OR

Toxicity Index is an integrated multiparametric toxicity assessment of each compound

concentration. The Toxicity Index is a weighted total (determined by the user) of three

nuclear features. Weights are assigned to each compound dose if it falls outside of user-defined thresholds for each feature. If a dose exceeds the final Toxicity Index threshold

Green (wells H1-H6) = Max Positive Control Yellow (wells H7-H12) = Vehicle Control Other = Sample Test Chemicals - range 100 µM

(row A) to 0.1 nM (row G) at 1:10 dilution in duplicate wells.


(also set by the user), it is flagged as toxic. The following features comprise the Toxicity

Index:

Cell Viability (Object Count) is calculated by measuring the number of nuclei

within a well.

Nuclear DNA Content (Nucleus Intensity) is monitored by measuring the total

integrated fluorescence intensity of the DNA dye (Hoechst 33342) in the nuclear

area of each cell with DNA staining.

Morphology (Nucleus Size) is monitored by measuring the nuclear size from

Hoechst-staining cells.

AND

Receptor Response measured through the following:

Receptor Foci Response is measured by integrating three different foci features (foci count, intensity, and area) in the nuclear area of each cell generated from the

GFP-expressing cells.

Receptor Translocation Response (AR only) is monitored by subtracting the

average cytoplasmic GFP fluorescence intensity from the average nuclear GFP

fluorescence intensity within each cell. In the AR assay, translocation is seen as the decrease of GFP from the cytoplasmic region and accumulation of GFP in the

nuclear region after treatment; the translocation event is not used in the ER Assay as most of the receptor resides in the nucleus of unstimulated cells.

Translocation is utilized as a secondary read-out for AR receptor activation.

Table 1. Assay Targets Used for the Dose Range Templates

TOXINSIGHT

IMAGING

CHANNEL

MONITORED CELL TARGET

CELLULAR REGION WHERE

MEASUREMENT IS MADE

(MEASUREMENT METHOD)

FLUORESCENT

PROBE COLOR

COMPARTMENTAL

ANALYSIS BIOAPPLICATION

OUTPUT FEATURE

1

Cell Viability Nucleus

(counting number of nuclei)

Hoechst 33342 Blue

ValidObjectCount

DNA Content Nucleus

(integrated intensity) ObjectTotalIntensityCh1

Morphology Nucleus

(nuclear size) NucSizeCh1

2

Foci Response

Foci formation in Cell Nuclear Region

(averaged from foci count, area, and intensity)

GFP tag Green

CircSpotTotalIntensityCh2

CircSpotTotalCountCh2

CircSpotTotalAreaCh2

Receptor Translocation

Response (AR only)

Cytoplasm to Nuclear Translocation

GFP tag Green CircRingAvgIntenDiffCh2


Receptor Activity Assays for both ER and AR

For the Receptor Activity Assays, six vials of CryoRedi cells are needed to test six compounds at two compounds per plate on duplicate plates (each compound run in triplicate per plate for

nine doses at 1:5 + the Maximum Positive Control concentration, a Positive Control dose

response, a Weak Response Control, and a Vehicle Control). Figure 6 represents the plate

layouts for this assay.

Figure 6. Suggested plate setup for Receptor Activation Plates. Six plates are needed per six compound set in triplicate wells, duplicate plates (2 sample test compounds and controls per plate). Compounds are added on top of plating media to give 1x final dilutions.

Plating, treating, and processing of the cells in the EPP Receptor Activity Assay is identical to the Dose Range Assay. The differences between the assays are the Sample compound‘s

starting concentration and dilution series, the plate layout, and additional controls in the

Activity Assays. For the Receptor Activity Assay, the following well types can be found:

1) Vehicle Control - VC (e.g., DMSO): used to normalize the data for nuclear

measurements and as a background response (lower limit or 0% response) for GFP

receptor measurements.

2) Max Positive Control - MaxPC (i.e., highest dose of -estradiol for ER or DHT for

AR): used to normalize the data on the maximal end for GFP responses and help

determine the thresholds for potency and potential mimic determinations; and which are

used to normalize the data for the GFP receptor responses (upper limit or 100% response) .

3) Positive Control - PC (i.e., dose response of -estradiol for ER or DHT for AR): used to

provide a dose dependent positive receptor response to compare response potency with

test compounds in addition to providing a positive response to the mimic determination.

4) Weak Response Control - WR (i.e., BPA for ER or progesterone for AR): used to

monitor positive foci responses to compare to the sample compounds.

5) Sample compounds - Sample: potential estrogenic/androgenic compounds that are being

assessed. Concentrations start at the dose determined from the Dose Range assays

followed by 8 additional 1:5 dilutions.

After treatment, the cells are fixed, washed and stained with Hoechst from the EPP

Cartridge(s) to simultaneously monitor the following cellular responses (Table 2):

Yellow (column 1) = Vehicle Control (1% DMSO) Blue, Red (wells A2-F10) = Sample Chemicals –

two compounds per plate; range determined from Dose Range plate at 1:5 dilutions in triplicate wells (A2-C2 and D2-F2 for highest dose).

Green (wells G2-H10) = Positive Control at 1:5 dilutions (starting at 50 nM β-estradiol or DHT)

Dark Green (column 11) = Max Positive Control (50 nM β-estradiol or DHT)

Purple (column 12) = Weak Response Control (500 nM BPA or 125 nM progesterone)


1) Cell Viability is calculated by measuring the number of nuclei normalized to the vehicle

control (set to 100% viability); a user-defined threshold flags compound concentrations

that decrease nuclei count below the threshold (default set to 80% viability).

2) Receptor Foci Response is measured by integrating three different foci features (foci

count, total foci intensity, and average foci area) in the nuclear area of each cell generated

from the GFP-expressing cells.

AND

3) Receptor Degradation (ER only) is measured by analyzing the integrated total GFP

fluorescence intensity in the nuclear area of each cell. Degradation is seen as the decrease

of GFP in the nuclear region after treatment.

OR

Receptor Translocation Response (AR only) is monitored by subtracting the average cytoplasmic GFP fluorescence intensity from the average nuclear GFP fluorescence

intensity within each cell. Translocation is seen as the decrease of GFP from the

cytoplasmic region and accumulation of GFP in the nuclear region after treatment.

Table 2. Assay Targets Used for the Receptor Activity Templates

Using Template Analysis to Make Receptor Response Predictions

for Potential Endocrine Disruption

There are two main templates for each assay that will differ in the importance of the features

along with the visualization and presentation of the data. For the Dose Range template, the

main output will monitor general toxicity with a secondary output of GFP receptor response (this plate determines the concentrations to be used in the Receptor Activity assays). For the

Receptor Activity template, the main output will analyze the GFP receptor foci response, a

secondary output for control similarity (potential mimic), and a tertiary output of toxicity. Following import of data into the templates, the responses are reported using a combination of

TOXINSIGHT

IMAGING

CHANNEL

MONITORED CELL TARGET

CELLULAR REGION WHERE

MEASUREMENT IS MADE

(MEASUREMENT METHOD)

FLUORESCENT

PROBE COLOR

COMPARTMENTAL

ANALYSIS BIOAPPLICATION

OUTPUT FEATURE

1 Cell Viability Nucleus

(counting number of nuclei) Hoechst 33342 Blue ValidObjectCount

2

Foci Response

Foci formation in Cell Nuclear Region

(averaged from foci count, area, and intensity)

GFP tag Green

CircSpotTotalIntensityCh2

CircSpotTotalCountCh2

CircSpotTotalAreaCh2

Receptor Degradation (ER)

Or Receptor

Translocation (AR)

Cell Nuclear region (integrated intensity)

OR Cytoplasm to Nuclear

Translocation

GFP tag Green CircTotalIntensityCh2

OR CircRingAvgIntenDiffCh2


user-defined inputs and thresholds formatted into the Microsoft Excel template. From the data,

the original purpose of the assay can be derived from and translated via the template to provide

endpoint predictions. Each type of template has the following purpose, endpoint, and outputs:

EPP Dose Range Assay and Template

Purpose: to determine a compound‘s starting dose for the Receptor Activity plate by

monitoring toxicity and the maximum receptor response

Endpoint: starting dose is defined as the minimum dose plus one dose that induces the

maximum receptor response and does not cause toxicity

Primary output: general toxicity assessment of each compound

Secondary output: GFP foci response to provide initial information on magnitude of the

receptor response for each compound

EPP Activity Assay and template

Purpose: to determine a compound‘s ability to induce a potential endocrine response by

measuring receptor activation

Endpoint: comparison of a compound‘s receptor dose response to the positive control

showing both the potency and magnitude of the response

Primary output: GFP foci response providing detailed information on both potency and

magnitude of the foci response for each compound

Secondary output: mimic determination comparing the magnitude of the sample compound‘s

response for two steps in the receptor activation pathway with respect to the positive control

Tertiary output: general information on the toxicity potential of the compounds

Using Quantitatively Imaged Data to Make the Proper Dose

Range Prediction

The EPP ER and AR templates have been designed to monitor both the receptor response

and toxicity following compound treatment by comparison to Vehicle and Max Positive Control sets. To accurately measure compound-induced receptor activity, it is first necessary

to determine the optimal dose range that captures a response curve in the absence of

deleterious cell effects. General toxicity is quantitated at the Well Level through multiple tunable outputs measuring nuclei count, nuclear size, and nuclear staining intensity to report

the toxicity of each dose. Toxicity thresholds can be set using ISO 10993-5:2009 [8] suggested

cut off of 80% cell viability or manually defined by a weighted Toxicity Index comprised of

values derived for nuclei count, size, and staining intensity.

In addition to toxicity evaluation, receptor response is measured though the formation of GFP

nuclear foci (monitoring foci size, foci intensity, and foci count per nuclei) and normalized to a

maximum response from the Max Positive Control. User-defined thresholds are utilized to

report the magnitude of the induced response from sample compounds compared to the Max Positive Control response. Plate acceptance criteria measuring data quality (Response

monitoring using fold- Max Positive Control induction and Vehicle Control background

monitoring and Max Positive and Vehicle Control Coefficient of Variation [CV] for measured


cellular features) are monitored and compared to lab standards ensuring proper assay function

and reproducibility.

The EPP Assay Templates utilize the quantitative data obtained from analysis of the imaged fluorescent cells to make the easily interpretable predictions by combining multiple cellular

responses. Data is imported from the ToxInsight Platform into the EPP Assay Microsoft Excel

Templates, which is then analyzed to predict receptor activation and toxicity. Analysis by the

EPP Dose Range templates is performed through the following outputs (see Figure 7):

1) Toxicity—chemical-induced cell death can have a dramatic impact on automated image

analysis of receptor response. The main output to determine toxicity is Cell Viability. An

additional option for a more detailed analysis of toxicity is the Toxicity Index (only

available on the Dose Range templates).

Cell Viability—this output monitors nuclei count. If a dose falls below a user-

defined threshold (default is set to 80%), it is flagged as toxic.

Toxicity Index (Dose Range template only)—this output is the weighted total of three

nuclear features: nuclei count, total nuclear fluorescence intensity, and nuclear size.

The Toxicity Index value is monitored for each dose by adding the weighted values of each individual feature. If a dose falls outside of the user defined thresholds for each

feature, the dose is flagged and assigned a weight (for example, if the weight for

nuclei count is 0.5, nuclear intensity is 0.3, and nuclei size is 0.2 and a dose is outside the thresholds for nuclei count and nuclei size, it is assigned a total value of 0.7). A

dose is flagged as toxic if it exceeds the user-defined Toxicity Index value. For the

example given above, if the Toxicity Index threshold is set to 0.6, the example dose

would exceed that threshold and would be considered toxic.

2) Receptor Response—this output is the average of three individual foci measurements:

intensity, area, and count.

Magnitude—the templates derive the amount of each sample‘s foci response in

comparison to the normalized positive control response (set to 100%) and reports two

outputs: flagged doses that exceed a threshold and the maximum response percentage.

Foci Response—this output reports whether or not a dose crosses a user- defined

threshold defined by the Positive Control Foci Response. (This data can also be

viewed using each individual foci measurement.)

%Foci Response—this output reports the maximum response of a compound

compared to the Positive Control response (which is set to 100%).


Compound

compound A %

ConcentrationObject

Count

Nucleus

Intensity

Nucleus

Size

Toxicity

IndexViability

Foci

Response

%Foci

ResponseDR Start

1E-04 - - + - - - 0.93%

0.001 - - - - - - 2.39%0.01 - - + - - - 1.06%

0.1 - - - - - - 2.80%

1 - - - - - + 93.48%

10 - - - - - + 152.01% Start

100 - - + - - + 75.43%

Figure 7. The EPP Dose Range templates couple toxicity data to receptor response data to generate a

suggested starting dose for the subsequent Receptor Activity assay.

From this information, a concentration will be suggested by the template to start the final

Receptor Activity experiments for these compounds. For ER , the suggesting starting dose is the concentration that is not toxic and gives the maximum receptor foci response plus one

dose. The ―plus one‖ dose stipulation ensures that the entire dose response is captured within the dilution series of the Activity Assay. If there is no receptor foci response, the starting dose

is the maximum non-toxic concentration. Examples of starting dose determinations are shown

in Figure 8.

For AR, the suggesting starting dose is the concentration that is not toxic and gives the

maximum receptor foci response plus one dose. If there is no receptor foci response, the starting dose is the concentration that is not toxic and gives the maximum receptor

translocation response plus one dose. It there is no receptor foci response or receptor

translocation, the starting dose is the maximum non-toxic concentration.

Toxicity measurements Response measurements


Thresholds

- - - - -

Compound - - - - -compound E

Concentration DR Start

1E-04

0.001

0.01

0.1

1

10

100 Start

Viability

Graphs

Foci Response

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

compound E

Thresholds

- - - - -



1E-04

0.001 Start

0.01

0.1

1

10

100

Viability

Graphs

Foci Response

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

compound E

No toxicity and no response above threshold = highest dose

No toxicity with minimum dose + 1 dose giving maximum response

Thresholds

- - - - -



1E-04

0.001

0.01

0.1

1

10

100 Start

Viability

Graphs

Foci Response

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

compound E

Thresholds

- - - - -



1E-04

0.001

0.01

0.1

1 Start

10

100

Viability

Graphs

Foci Response

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

compound E

No toxicity with maximum dose giving maximum response Highest non-toxic dose giving maximum response

Thresholds

- - - - -

Compound - - - - -compound A


1E-04

0.001

0.01

0.1

1

10 Start

100

Viability

Graphs

Foci Response

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

compound A

Thresholds

- - - - -

Compound - - - - -compound C


1E-04

0.001

0.01

0.1 Start

1

10

100

Viability

Graphs

Foci Response

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

compound C



Figure 8. Examples of starting dose selection. The Dose Range templates analyze toxicity and response data to suggest a

starting dose for the Receptor Activity Assay. The yellow arrow on each graph points to the selected starting dose with the logic used in selecting that dose below the graph.


Using Quantitatively Imaged Data to Evaluate Receptor Activity

For the second set of templates, the magnitude and potency of the receptor foci response are the main output values. However, toxicity thresholds are also taken into account (only by using

ISO 10993-5:2009 [8] suggested cut off of 80% cell viability). Toxicity within a well is

controlled at the cellular level by using nuclei selection and GFP-receptor gating criteria to

exclude dead/abnormal cells from analysis giving more consistent receptor response data Please refer to Chapter 4 and the Compartmental Analysis BioApplication Guide for additional

information on object selection and gating.

As performed in the Dose Range template, receptor response is measured though the formation

of GFP nuclear foci (monitoring foci size, intensity, and count per nucleus) and normalized to a maximum response from the Positive Control. User-defined thresholds are utilized to report

both the potency of test compounds compared to the Max Positive Control dose response

(expressed as the concentration that a dose response crosses a defined threshold) and the magnitude of the induced receptor response (expressed as a percentage of the maximum

positive control response). A Weak Response Control is included in the Receptor Activity

Assays to provide an additional positive receptor response. Plate acceptance criteria measuring

data quality are monitored and compared to standards ensuring consistent assay function and

reproducibility.

An additional aspect that is incorporated into the Activity assays is the ―mimic‖ potential for

each compound. The mimic feature will monitor a test compound's ability to imitate the

Positive Control by evaluating if the test compound responds in a similar way to two receptor response features. This analysis provides insight into the compound‘s mechanism of action

during receptor activation and signifies that further investigation of the compound‘s potential

for endocrine disruption may be warranted. Both ERα and AR assays monitor unique

potential mimic features and are described in detail below.

The EPP templates utilize the quantitative data obtained from analysis of the imaged fluorescent cells to make the easily interpretable predictions by combining multiple cellular

responses. Data is imported from the Store database (via the Thermo Scientific iView

software) into the EPP Assay Analysis Microsoft Excel Templates, which is then analyzed to

predict potential receptor activation, toxicity, and mimic functionality.

Analysis by the EPP Receptor Activity templates is performed through the following major

outputs (Figure 9):

1) Toxicity (Viability) - chemical-induced cell death can have a dramatic impact on

automated image analysis of receptor response. This is calculated as in the Dose Range templates as Viability to monitor nuclei count. If a dose fails below a user-defined

threshold (default is set to 80%), it is flagged as toxic.

2) Receptor Response - this output is the average of three individual foci measurements:

intensity, area, and count.

Magnitude - the templates derive the amount of each sample‘s foci response in

comparison to the normalized Max Positive Control response (set to 100%) and

reports two outputs: flagged compounds that exceed a threshold and the maximum

response percentage.

Foci Response—this reports whether or not a dose crosses a user- defined threshold defined by the Max Positive Control Foci Response. This data can also

be viewed using each individual foci measurement. There are three user-defined


thresholds calculated against the Max Positive Control (P1, P3 and P3, set to

defaults of 20%, 50%, and 100%, respectively) as well as evaluation against the

Weak Response Control.

%Foci Response—this reports the maximum response of a compound compared

to the Max Positive Control response (which is set to 100%).

Potency (Activity templates only) - linear regression is used to interpolate the

concentration at which sample dose responses cross user-defined thresholds. There

are three user-defined thresholds calculated against the Max Positive Control (P1, P3 and P3, set to defaults of 20%, 50%, and 100%, respectively) as well as evaluation

against the Weak Response Control (WR threshold). Linear regression analysis using

the equation x = (y – b) / m (where x is the exact concentration that the threshold, y is the threshold, b is the y-intercept, and m is the line slope) is performed on the line

between the concentrations that cross the threshold (the last concentration below and

the first concentration above the threshold).

For sample compounds, a Max Positive Control threshold set at 50% is not equivalent to the EC50 value of the compound and should not be utilized as such.

3) Potential Mimic (Activity template only)—this output compares two distinct steps in the

receptor activation pathway to determine if a sample-induced response is imitating the response of the Positive Control. The mimic determination is different for each EPP cell

line:

ER : Receptor degradation (GFP nuclear intensity) and foci response will be used as

the potential mimic feature. For example, -estradiol (the ER natural ligand and the

ER assay positive control) induces nuclear ER foci formation followed by receptor

degradation to dampen/end the response.

AR: For AR, nuclear translocation of the receptor (translocation) and foci response will be used as the potential mimic feature. For example, DHT (the AR natural ligand

and the AR assay positive control) induces nuclear translocation followed by nuclear

foci formation.

Comparison of the response thresholds (P1, P2, and P3) that are flagged for both foci response and the potential mimic feature will determine if the samples exhibit similar

receptor activity, giving three possible outcomes (Max Positive Control responses for both

foci and degradation/translocation set to 100%):

1) No Response (N/R) - this signals that the sample compound does not induce

either of the responses above the defined thresholds.

2) Mimic—this signals that the sample compound is similar to the positive control and crosses the same thresholds for both responses. For example, a compound is

considered a potential mimic in the ER assay if the responses exceed the P1 and

P2 but not P3 thresholds for both foci response and receptor degradation.

3) No Mimic—this signals that the sample response is different for the measured

features. For example, a compound is considered not a mimic in the ER assay if the responses exceed the P1 and P2 and not P3 thresholds for the Foci Response

but does not exceed any thresholds for receptor degradation. The failure to


promote receptor degradation could be due to a number of causes including

inhibition of receptor phosphorylation preventing proteosome recognition or the formation of unproductive nuclear foci [9]. Alternatively, a compound response

that does not exceed any thresholds for Foci Response but elicits receptor

degradation exceeding P1 and P2 thresholds is also not a mimic as it promotes

receptor degradation in the absence of activation. For the AR assay, similar situations such as exceeded thresholds for nuclear translocation in the absence of

exceeded Foci Response thresholds suggest that the compound fails to induce foci

formation after translocation. This data could provide doses or compounds that

bind AR but do not activate AR-mediated gene transcription.

The threshold levels determine the final analysis for both the foci response and potential mimic functions. Setting a threshold below the background response level may result in false positives and incorrect mimic designations. See Assessing Plate Data Quality (Chapter 3, Step 2) for monitoring background responses.

Foci Response %

UniquePlateID Compound Viability P1 WR P1 P2 P3 % Foci Response Line

USPI2-SWTEST24_110520090002 b-estradiol - + Mimic 0.00006 0.00008 0.00024 0.00041 109.90%


USPI2-SWTEST24_110520090002 compound A - + No Mimic 0.31632 0.41951 0.69481 0.97012 192.23%


USPI2-SWTEST24_110520090002 compound B - - No Mimic #N/A #N/A #N/A #N/A 11.02%

USPI2-SWTEST24_110520090001 compound B - - No Mimic 9.25110 #N/A #N/A #N/A 14.12%

Potential

Mimic

Figure 9. The EPP Receptor Activity templates generate detailed analysis of potency and magnitude of

receptor responses to assess a compound‟s potential for endocrine disruption in addition to comparing compounds to the positive control (Potential Mimic).

Stepwise Extrapolation of Response Predictions for both Dose

Range and Receptor Activity Templates

The EPP templates perform the following steps to extrapolate response predictions from

cellular data:

1) Data Normalization using Vehicle and Positive Controls

2) Assessing Plate Data Quality and Reviewing Plate Acceptance Criteria

3) Determining Thresholds

4) Identifying Toxicity Responses for Individual Targets

5) Identifying Foci Receptor Responses for Individual Targets

6) Applying Thresholds to Determine Compound Concentration from Dose Range

Templates

7) Applying Thresholds to Determine Receptor Response Potency and Magnitude from

Receptor Activity Templates

8) Applying Thresholds to Determine Potential Mimics from Receptor Activity Templates

Potency determinations

Magnitude determinations


Step 1. Data Normalization using Vehicle and Positive Controls

The first step in the analysis is that the raw data for each well and each measured feature (from the Rawdata tab) are normalized using the values from the Vehicle and Max Positive Control-

treated cells (found on the Basal Response tab). The normalized data is reported on the

Normalized Data tab. Normalization is performed on a plate-by-plate basis when multiple plates are imported (only available in Activity templates) enabling direct comparisons between

the different plates within a single template. Table 3 is an overview of the calculations that

consist of:

Raw data for nuclear features are normalized versus the mean of the Vehicle Control

measurements.

Raw data for GFP Receptor measurements (with the exception of GFP Nuclei Total Intensity) are normalized first by subtracting the baseline mean of the Vehicle Control

before dividing by the mean Max Positive Control response above baseline.

Raw data for GFP Nuclei Total Intensity is normalized first by subtracting the lower

response limit (mean of the Max Positive Control) before dividing by the baseline

mean Vehicle Control value above the mean Max Positive Control response.

Table 3. Calculations used to normalize data. VC = Vehicle Control, MPC = Max Positive

Control

Feature Normalization Calculation

Nuclei features (count, size, intensity) Rawdata / VCmean

GFP response (foci area, intensity,

count, translocation)

(Rawdata – VCmean) / ( MPCmean – VCmean)

GFP Nuclei Total Intensity (Rawdata – MPCmean) / ( VCmean – MPCmean)

Step 2. Assessing Plate Data Quality and Reviewing Plate

Acceptance Criteria

The EPP templates feature a data quality assessment tool that automatically compares the raw data of control compounds to set thresholds in order to demonstrate system performance

continuity. The Basal Response tab of each template contains a detailed table that monitors

variability within the control measurements and the background and fold induction of the Foci

Response (Figure 10); a summary of these measurements assessing overall plate health is reported on the Conclusions tab to couple plate acceptance and response quality with final

data analysis (Figure 11). Data quality and acceptance criteria are analyzed and reported on a

per plate basis (each plate is analyzed individually). Criteria that are within the defined limits are designated as ―Pass‖ and have data quality that meets or exceeds the default limits.

Measurements that do not meet the defined quality criteria are assessed as ―Verify.‖ This

designation suggests that the data on this plate exhibits higher than specified variability. It should be noted that a ―Verify‖ designation does not automatically eliminate the plate from

further analysis. Investigation of the variation by utilizing the QC tab to identify outlier wells

and inspecting of the cells and images is suggested to ascertain the cause of the poor data

quality.


Data Quality is assessed using the following criteria:

1) Vehicle Control—the CV (Coefficient of Variation) is calculated from the mean and

standard deviation for each nuclear measurement (count, intensity, and size) and the GFP

intensity (total nuclear GFP intensity for ER and translocation for AR) and compared to a pre-defined CV threshold. Low variation within the Vehicle Control nuclear features

ensures good cell health while low variation within the nuclear GFP intensity

demonstrates consistent expression of the tagged receptor.

2) Max Positive Control—In addition to the calculations described above, the CV for each

foci measurement (count, intensity, and area) is compared to pre-defined thresholds to

demonstrate consistent Foci Responses in the Max Positive Control.

3) Foci Response— A very important metric for assessing data quality when utilizing

threshold-based analysis is monitoring both the background level and fold induction of the

response. The background level assessment for each foci response (count, intensity, area)

is derived from the minimum receptor response for the Max Positive Control (default set at 20% of the mean) that should be above the variation of the Vehicle Control baseline

response (default set at the mean of the Vehicle Control plus 2.5 x standard deviation

[VCmean + 2.5(VCSD)]). Responses in which 20% of the Max Positive Control exceed the variation in the baseline Vehicle control (2.5 x standard deviation represents >99% of the

variation) are designated as ―Pass.‖ Background measurements that do not meet this level

are assessed as ―Verify‖ suggesting that the data on this plate exhibits higher than

specified variability and setting a response threshold at or lower than 20% may result in an

increase in false positives.

The fold induction describes the magnitude of the Max Positive Control response

compared to the Vehicle Control baseline response; the greater the fold induction, the

greater the separation between the maximum and minimum responses resulting in a more accurate assessment of values in between. For the EPP assays, the fold induction limit of

the receptor response for each foci feature (count, intensity, area) is monitored by

comparing the mean of the Vehicle Control (VC) multiplied by the VC multiplier (default

set at 10 for a 10-fold induction) to the mean of the Maximum Positive Control (MPC) multiplied by the MPC multiplier (default set to 1). Responses that exceed a 10-fold

induction are designated as ―Pass‖ whereas those that are less than 10-fold are flagged by

―Verify.‖ An assessment of ―Verify‖ suggests either a variable Max Positive Control response or a high Vehicle Control baseline response and caution should be used when

interpreting the results.


WellType Feature Mean SD CV CV Threshold Quality

Vehicle Object Count 245.1667 18.9886 0.0775 0.15 Pass

Nucleus Intensity 305141.8239 12347.6785 0.0405 0.15 Pass

Nucleus Size 31.4272 0.4162 0.0132 0.15 Pass

GFP Nucleus Intensity 285353.5447 15364.4495 0.0538 0.30 Pass

WellType Feature Mean SD CV CV Threshold QualityMax Positive Object Count 248.6667 38.0111 0.1529 0.15 Verify

Nucleus Intensity 317783.7568 12636.8225 0.0398 0.15 Pass

Nucleus Size 32.2596 0.2924 0.0091 0.15 Pass

GFP Nucleus Intensity 216137.4037 8912.6820 0.0412 0.30 Pass

Foci Intensity 7767.3704 908.2623 0.1169 0.30 Pass

Foci Area 1.8680 0.0760 0.0407 0.30 Pass

Foci Count 4.3573 0.3970 0.0911 0.30 Pass

Response Feature MPC * Multiplier VC + SD*Multiplier MPC MultiplierVC Multiplier QualityBackground Foci Intensity 1553.4741 212.9908 0.20 2.5 Pass

Foci Area 0.3736 0.1454 0.20 2.5 Pass

Foci Count 0.8715 0.2248 0.20 2.5 Pass

Response Feature MPC * Multiplier VC * Multiplier MPC MultiplierVC Multiplier QualityFold Induction Foci Intensity 7767.3704 1145.6621 1.00 10 Pass

Foci Area 1.8680 0.7602 1.00 10 Pass

Foci Count 4.3573 1.1976 1.00 10 Pass

Figure 10. Dose Range template: Basal Response Tab – Visualization of plate acceptance criteria and data quality assessment. For Activity templates, each plate has a corresponding acceptance criteria table.

Results from the data quality assessment are summarized on the Conclusions tab as Plate

Acceptance Criteria (Figure 11). If all the measurements meet the acceptance criteria, the plate acceptance is designated as ―Pass.‖ If one of the parameters does not meet the acceptance

criteria, the plate is assigned a Plate Acceptance output of ―Verify.‖ The final judgment on

accepting plates for analysis is the responsibility of the user.

Quality

Pass

Pass

Pass

USPI2-SWTEST24_110518140001

Vehicle

Max Positive

Response

Figure 11. Dose Range template: Conclusion Tab – Visualization of summarized data quality assessment. For Activity templates, each plate has a corresponding Plate Acceptance Criteria table.

Step 3. Determining Thresholds

The EPP Assay deals with the issue of non-unidirectional or non-classic concentration-

response curves by defining thresholds for each of the measured cellular targets and flagging

the compound if any of the targets go beyond the threshold for each concentration tested. Determining the toxicity thresholds is a critical step for this assay, as it deals with the

variations in specific target responses over a range of concentrations and improves the

robustness of the assay‘s predictions.

The thresholds for each of the measurements are determined from the normalized mean

response of the Vehicle Control and/or the Max Positive Control. For each of the features, the mean and standard deviation of the normalized response of the Vehicle and Max Positive

Control compounds are calculated (this data is shown on the Normalized Response tab of the

EPP templates).


Two thresholds (upper and lower) for each target measured (mean plus and mean minus the

user-defined coefficient multiplied by the standard deviation) are utilized for the following

features:

Nuclear intensity (Dose Range templates only)

Nuclear size (Dose Range templates only)

Threshold = Normalized Mean ± (Coefficient × Standard Deviation)

Lower thresholds are used to monitor changes in cell number as cell number increase

(proliferation) is not considered to be a toxic response:

Nuclei Count (Dose Range templates only)

Threshold = Normalized Mean – (Coefficient × Standard Deviation)

To set the threshold for the Weak Response Control foci response (WR), only the upper limit

is set using the normalized mean plus the standard deviation:

Foci Response for Weak Response Control (WR; Receptor Activity templates only)

Threshold = Normalized Mean + (Coefficient × Standard Deviation)

For certain cellular targets, where it makes biological sense, thresholds are based on a

percentage of the normalized response (user-defined value expressed as a decimal) of the

Vehicle or Max Positive control. For these features, doses with values that exceed or fall

beneath the threshold are flagged as a response.

The following features use only the upper threshold:

Foci Response

Foci features (count, intensity, and area)

Positive Control thresholds (P1, P2, and P3; Receptor Activity only)

Translocation (AR Dose Range only)

Threshold = Normalized Mean × User-defined Value

The following features use only the lower threshold:

Viability

Threshold = Normalized Mean × User-defined Value

For determination of the Toxicity Index, the threshold is simply a user-defined value

(percentage expressed as a decimal). If a dose exceeds this value, it is flagged as a toxic dose:

Toxicity Index (Dose Range only)

Threshold = User-defined Value


Determining and setting threshold levels is an important aspect of this assay and should take into account plate control variation, background, and fold induction (See Step 2: Assessing Plate Data Quality and Reviewing Plate Acceptance Criteria), individual requirements for positive responses, and/or empirically derived levels that are biologically relevant. It is important to consider these factors when determining the thresholds as data analysis and interpretation can be severely impacted by aberrant threshold levels leading to potential increased false positive/negative determination and inconsistent analysis.

Step 4. Identifying Toxicity Response for Individual Targets

The toxicity response against a sample compound is evaluated for each dose over the full

concentration range of the sample compound. If the normalized response for a particular dose

is beyond the toxicity thresholds, the compound is flagged as giving a toxic response. For the Dose Range templates, toxicity response values are used in a dose-by-dose manner to

determine the starting dose range for the Activity assay. There are two separate means for

determining toxicity: (1) Viability and (2) Toxicity Index.

Cell Viability – This is based on the actual number of cells counted within the well (percent expressed as a decimal) that is normalized to the mean of the Vehicle Control. Any values that

fall beneath the lower limit (representing a decrease in cell number) are flagged as positive

(Figure 12A).

Toxicity Index – This is a weighted combination of Object Count, Nuclear Intensity, and

Nuclear Size (three features connected with early and late stages of toxicity). Figure 12B shows an example of how the Toxicity Index is determined. You can determine the

importance of each feature by applying different ‗weights‘ (i.e., in the example, Object Count

is weighted at 0.2, Nuclear Intensity at 0.5, and Size at 0.3). For concentrations of a compound that exceed the individual thresholds for any of the three features, the index value will be

determined by summing the weighted values for the feature(s) that exceed the threshold. If the

accumulated weight is greater than the Toxicity Index threshold that you set (i.e. Toxicity

Index threshold set at 0.55 in the example), the concentration is flagged as positive. In the example, the compound in red has a dose with a 1.0 Toxicity Index (0.2 from Object Count +

0.5 from Nucleus intensity + 0.3 from Nucleus Size). Both the light blue and orange

compounds also have toxic doses with 0.8 Toxicity Index values (0.0 from Object Count + 0.5

from Nucleus intensity + 0.3 from Nucleus Size).

The choice and adjustments of toxicity determination methods (Viability or Toxicity Index)

can impact the final analysis of the samples and the suggested starting dose for the Activity

assays. In the example shown in Figure 12 which shows the same set of compounds, assessing toxicity via the Toxicity Index results in concentrations of the light blue and orange lines that

are considered toxic and would be eliminated as potential starting doses. The same

concentrations from these compounds are not flagged as toxic using Viability and would be

used in starting dose prediction.


- - -

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Viability

Foci Response

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Object Count

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Nucleus Intensity

0.8800

0.9000

0.9200

0.9400

0.9600

0.9800

1.0000

1.0200

1.0400

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Nucleus Size

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Toxicity Index

Viability

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Object Count

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Nucleus Intensity

0.8800

0.9000

0.9200

0.9400

0.9600

0.9800

1.0000

1.0200

1.0400

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Nucleus Size

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Toxicity Index

Viability

Figure 12. Example of toxicity threshold determination using 2 different methods. (A) Cell Viability calculated for six separate compounds. Threshold levels are shown to the right. Four of the six are above the set threshold, indicating that viability has not been affected. The remaining two compounds

(red and green lines) show a drop in viability and would be flagged as positive. (B) Toxicity Index calculated for the same six compounds. Taking into account object count, nuclear intensity and nuclear size (early and late stages of toxicity), different compounds are now „flagged‟ as positive with three

of the six compounds exceeding the set threshold (red, light blue, and orange lines).

A

B


Step 5. Identifying Foci Receptor Responses for Individual

Targets

The Foci Response (found in both Dose Range and Receptor Activity templates) graphs the average of three separate features: Foci Count, Intensity, and Area that are normalized to the

Maximum Positive Control response. Individual thresholds are set for each feature and are

replicated throughout all 4 graphs (i.e., if 20% [0.2] is used for the threshold, it is 0.2 for all four graphs), providing a consistent threshold for comparing responses for each output.

Figure 13 is an example of compounds showing similar graphs for Foci Response, Intensity,

Area, and Count. However, this may not be the case if there are experimental artifacts (i.e.,

toxic nuclei with Hoechst bleed-through into the GFP channel or fluorescent debris that may drastically increase Foci Intensity, but not the Foci Count and/or Area; thus the overall Foci

Response will be averaged accordingly). For the AR Dose Range template, thresholds are

utilized for receptor translocation in a manner similar to the Foci Response threshold (percent

expressed as a decimal, normalized as Rawdata-VCmean/MPCmean-VCmean).

0.0000

0.5000

1.0000

1.5000

2.0000

2.5000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Foci Intensity

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.4000

1.6000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Foci Area

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.4000

1.6000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Foci Count

Concentration

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.4000

1.6000

1.8000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Foci Response

× SD Weight Threshold ThresholdObject Count 2 0.2 Viability 75.00% Response 20.00%

Nucleus Intensity 1 0.5 Intensity 20.00%

Nucleus Size 1.5 0.3 Area 20.00%

Threshold Count 20.00%

Toxicity Index 0.55

Viability Foci ResponseToxicity Index

0.0000

0.5000

1.0000

1.5000

2.0000

2.5000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Foci Intensity

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.4000

1.6000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Foci Area

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.4000

1.6000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Foci Count

Concentration

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.4000

1.6000

1.8000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Foci Response

Figure 13. Example of Foci Response and the three features that make up this graph. The threshold for the control (user-adjustable in yellow) represents the thresholds set in each individual graph.

Step 6. Applying Thresholds to Determine Compound

Concentration from Dose Range Templates

Graphs Tab

To determine the appropriate starting dose to use for dilution series in the Receptor Activity

assay, you must apply appropriate thresholds for the Foci Receptor Response feature(s) in

addition to either of the modes of toxicity assessment (either Viability or Toxicity Index).

For each template, thresholds are adjusted in the Graphs tab. The values of the individual thresholds are calculated and reported in other template tabs but they can only be adjusted in the Graphs tab.


Thresholds for individual components of the Toxicity Index are initially calculated from

Vehicle Control values of the individual nuclei measurements and further from the combined

weight of these features (Figure 12).

Viability (Vehicle Control Nuclei Count) and Foci Response (normalized Max Positive

Control response) are set off of a percent threshold (expressed as a decimal) and flag values

falling beneath a threshold (decrease in Viability) or exceeding the threshold (increase in Foci

Response). Adjusting these values will adjust the threshold lines within the appropriate graphs

(Figures 12 and 13).

Figure 14 is an example of the features and those that you can alter (in yellow). There are a

total of nine graphs for the Dose Range template for the ER assay (Figure 15), and ten graphs

for the Dose Range template for the AR Assay.

× SD Weight Threshold ThresholdObject Count 2 0.4 Viability 80.00% Response 20.00%

Nucleus Intensity 2 0.3 Intensity 20.00%

Nucleus Size 2 0.3 Area 20.00%

Threshold Count 20.00%

Toxicity Index 0.5

Viability Foci ResponseToxicity Index

Figure 14. Thresholds available to set in the ER Dose Range template are highlighted in yellow. In the AR

Dose Range template, an additional threshold for Translocation will also be present.

- - - Viability Threshold

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Object Count

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Nucleus Intensity

0.8800

0.9000

0.9200

0.9400

0.9600

0.9800

1.0000

1.0200

1.0400

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Nucleus Size

0.0000

0.5000

1.0000

1.5000

2.0000

2.5000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Foci Intensity

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.4000

1.6000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Foci Area

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.4000

1.6000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Foci Count

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Toxicity Index

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Viability

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.4000

1.6000

1.8000

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

Foci Response

Figure 15. Graphs Tab – Example of graphs available for the ERα Dose Range template. Note that for the AR Dose Range Template, a 10

th graph (translocation) will also be present.


Once all thresholds are set, each compound that has any concentration exceeding the set thresholds are noted as flagged (red, +). If a compound did not cross a threshold at any

concentration, it will not be flagged (green, -). Figure 16 is an example of the available features

showing both individual outputs (Object Count, Nuclei Intensity, Nuclei Size, Foci Intensity,

Foci Area, and Foci Count) and measurements that can be used to determine the starting dose (Toxicity Index, Viability, and Foci Response). In the first two yellow columns, the

compounds can be adjusted as well as whether the mean or individual replicates are graphed

and monitored for flagged responses. For the Dose Range Assay, each compound dilution is performed in duplicate; therefore, you can look at the first replicate, the second replicate, or the

mean calculated from both wells. The responses from the mean and each replicate can also be

compared by selecting the same compound in different rows along with different replicates

selected in the replicates column.

Compound RepLine

Color

Object

Count

Nucleus

Intensity

Nucleus

Size

Foci

IntensityFoci Area Foci Count

Toxicity

IndexViability

Foci

Response

1 compound A Mean - - + + + + - - +

2 compound B Mean - - + - - - - - -

3 compound C Mean + - - - - - - + -

4 compound D Mean - + + - - - - - -

5 compound E Mean - - - - - - - - -

6 compound F Mean + - + + + + - + +

7 compound F 2 + - + + + + - + +

8 compound F 1 + + + + + + + + +

Figure 16. Overall Assay Table showing the available features, if they are „flagged‟, as well as the adjustable

appearance of the graphs (compounds and replicates, in yellow). Features that can be used to determine the starting dose concentration are Toxicity Index or Viability and Foci Response. The AR Dose Range template has an additional column to the right of the Foci Response to monitor Translocation.

Conclusion Tab

The main function of the Conclusions tab of the Dose Range templates is to determine the starting concentration for the subsequent Activity Assay. Also in the Conclusions tab, the

calculated and/or defined thresholds values are reported (Figure 17) but cannot be altered

(changing threshold values can only be performed in the Graphs tab). Compounds are selected and displayed one at a time to determine the concentration to select for starting the

compound dilutions for the Receptor Activity Assay (Figure 18).

Results for a toxicity measurement (either Toxicity Index or Viability) and the Foci Response

are graphed to the right of the table. The percentage of the Foci Response for each dose

compared to the normalized Maximum Positive Control response (set to 100%) is determined from the graph and reported in the % Foci Response column. The %Foci Response represents

the magnitude of the induced receptor foci response (responses can exceed 100% of the Max

Positive Control response). The starting dose is determined by integrating the toxicity measurement with both of the Foci Response outputs. Either Toxicity Index or Viability can

be used to determine toxicity (the method that is calculated is determined by selecting the

method above the graph; Figure 19). Changing the type of toxicity assessment may result in different suggested starting concentrations as each method is dependent on specific nuclear

measurements and thresholds. It is recommended that if there are discrepancies between the

two toxicity measurements, assessment of original images (in iView™ software) should be

used to help decide which concentration to use for the Receptor Activity Assay (i.e., small, condensed nuclei that are counted but do not decrease Viability and result in a higher starting

dose may result in a higher Toxicity Index and lower starting dose). For AR, %Translocation

(determined from the Max Positive Control response set to 100%) is also graphed and used to determine the starting dose for the Activity template if there is not a foci response that

exceeded the foci response threshold.


Object

Count

Nucleus

Intensity

Nucleus

Size

Toxicity

IndexViability

Foci

ResponseWeight 0.40 0.30 0.30

Threshold Min 0.8451 0.9191 0.9735 80.00%

Threshold Max 1.0809 1.0265 0.50 20.00%

Figure 17. Conclusion Tab showing threshold values from the ER Dose Range template - Review of weights

and thresholds selected in the Graphs Tab

Compound

compound F %

ConcentrationObject

Count

Nucleus

Intensity

Nucleus

Size

Toxicity

IndexViability

Foci

Response

%Foci

ResponseDR Start

1E-04 - - - - - - 2.83%

0.001 - - - - - - 1.68%

0.01 - - - - - + 59.11%

0.1 - - - - - + 125.10%1 - - - - - + 124.62% Start

10 + + + + + + 141.43%

100 + - + + + + 160.65%

Figure 18. Conclusion Tab from ERα Dose Range Template - Analysis of Toxicity and Foci Response. Selection of one compound showing individual concentrations and its effects on each feature as well as a review of the potency of the receptor and a suggestion of which concentration to start the Receptor Activity

Assay.

Thresholds

- - - - -

- - - - -

Viability

Graphs

Foci Response

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

compound F

Thresholds

- - - - -

- - - - -

Toxicity Index

Graphs

Foci Response

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

1.00E-04 1.00E-02 1.00E+00 1.00E+02

Concentration

compound F

Figure 19. Conclusion Tab from ER Dose Range template - Graphs representing Viability (A) and Toxicity

Index (B)

B A


Step 7. Applying Thresholds to Determine Receptor Response

Potency and Magnitude from Receptor Activity Templates

Graphs Tab

The Receptor Activity template Graphs tab uses the Viability thresholds as the individual

measurement of toxicity. The Activity templates also provide three adjustable thresholds based off of the Max Positive Control response (percentage for P1, P2, and P3) and a fourth threshold

derived from the Weak Response Control (WR, standard deviation) (Figure 20). Adjustments

to each threshold changes the levels on each Receptor Response graph (Foci Response, Foci

Intensity, Foci, Area, Foci Count; and Nuclear GFP Total Intensity for ER or Translocation for AR). The multiple thresholds provide an easy visual assessment of the magnitude of the

responses and will be used in the Conclusions tab to provide potency calculations.

Threshold Threshold

Viability 80.00% P1 20.00%

P2 50.00%

P3 100.00%

SD

WR 1

Viability Foci Response

Figure 20. Thresholds available to set in the Receptor Activity template within the Graphs tab

Values for each threshold can be found in the Normalized Response worksheet.

For the WR threshold, the value is derived from the WRmean + WRSD x User-defined Coefficient; therefore, the WR thresholds will vary between each of the foci measurements (intensity, area, and count). This threshold is meant to provide a guide for the response level for a compound that elicits a weak response.

Again, once all thresholds are set, any compound that has any concentration exceeding the set thresholds are noted as flagged (red, +) in both the Graphs and Conclusion tabs. If a

compound did not cross a threshold at any concentration, it will not be flagged (green, -).

Visualization of the flagged Foci Responses for Foci Intensity, Area, and Count in the results

table is controlled by adjusting the individual foci feature menu located at the top of the table. Figure 21 provides an example of the available graphs in the Activity template. For the ER

Assay, a graph of GFP Total Intensity within the nuclear region will be present. For the AR

Assay, GFP receptor translocation (from cytoplasm to nucleus) will be featured instead. As with the Dose Response template, the compounds can be selected as well as whether the mean

or individual replicates are graphed and monitored for flagged responses (Figure 22). For the

Activity assay, each compound dilution is performed in triplicate and the Positive Control in

duplicate; therefore, you can look at each replicate or the mean value. The responses from the mean and each replicate can also be compared by selecting the same compound in different

rows along with different replicates in the replicates column. In addition, multiple imported

plates (up to 3) can be graphed simultaneously to view consistency between plates.


Figure 21. Graphs Tab – Example of graphs available for the ER Receptor Activity template. Note that

for the AR Receptor Activity Template, GFP nuclear intensity will be replaced with Translocation.

UniquePlateID Compound RepLine

ColorViability WR P1 P2 P3 P1 P2 P3

USPI2-SWTEST24_110520090002 b-estradiol Mean - + + + + + + +

USPI2-SWTEST24_110520090001 b-estradiol Mean - + + + + + + +

USPI2-SWTEST24_110520090002 compound A Mean - + + + + + - -

USPI2-SWTEST24_110520090001 compound A Mean - + + + + + - -

USPI2-SWTEST24_110520090002 compound B Mean - - - - - + - -

USPI2-SWTEST24_110520090001 compound B Mean - - - - - + - -

USPI2-SWTEST24_110520090001 compound B 3 - - - - - + - -

USPI2-SWTEST24_110520090001 compound B 1 - - - - - + + -

Foci Response GFP Nucleus Intensity

Figure 22. Graphs Tab - Overall Assay Table showing the available features, if they are „flagged‟, as well as

the adjustable appearance of the graphs (compounds and replicates, in yellow).

Conclusion Tab

The main function of the Conclusions tab of the Activity templates is a detailed analysis of a

compound‘s receptor response in comparison to the Max Positive Control and the Positive

Control response curve, providing a finalized report of the receptor responses. In this tab, the calculated and/or defined thresholds values are reported (Figure 23) but cannot be altered

(changing threshold values can only be performed in the Graphs tab).

Figure 24 shows the Conclusions tab of the Activity templates which summarizes the toxicity

data (Viability column), provides final assessment of the receptor foci response (Foci

Response column), and determines potential mimic functionality (Potential Mimic column, see Step 8 for details). The determination of Foci Response can be based on any of the four pre-

defined thresholds (P1, P2, P3, and WR). Additionally, linear regression analysis is used to

interpolate the concentration at which the response crosses each threshold and comparison with the results from the Positive Control response provides the potency of the compound.

Finally, the maximum induced Foci Response for each compound (magnitude) is reported as

the %Foci Response. The graph shown in the Conclusions tab is a copy of the Foci Response

found in the Graphs tab and provides visual assessment and reporting of the main receptor response (Figure 25). Data from multiple plates can be displayed simultaneously to assess

plate-to-plate variability (select the plates using the Plate ID column and the compounds from

the compound column).


Viability Foci Response

Threshold Min 80.00%

Threshold WR 1.0Threshold P1 20.00%

Threshold P2 50.00%

Threshold P3 100.00%

Figure 23. Conclusion Tab of Activity template - Review of weights and thresholds selected from the Graphs

Tab. No editing can be done in this location.

Foci Response %

UniquePlateID Compound Viability P1 WR P1 P2 P3 % Foci Response Line





USPI2-SWTEST24_110520090002 compound B - - No Mimic #N/A #N/A #N/A #N/A 11.02%

USPI2-SWTEST24_110520090001 compound B - - No Mimic 9.25110 #N/A #N/A #N/A 14.12%

Potential

Mimic

Figure 24. Conclusion Tab of Activity template – Calculation of values determined from set thresholds as

well as potency compared to the Max Positive Control and Positive Control dose response.

Figure 25. Conclusions Tab of Activity template – Overview of the Foci Response for compounds selected for analysis.

Step 8. Applying Thresholds to Determine Potential Mimics from

Receptor Activity Templates

The mimic determination within the Receptor Activity templates tests the similarity between

two responses in the receptor pathway for each compound with respect to the Max Positive

Control (MPC). By definition, each Foci Response for the MPC response is set to 100%; therefore, the MPC gives similar responses to each feature. To be considered a potential

mimic, a compound must give similar responses for each feature. Responses are compared

using threshold crossing set with the P1, P2, and P3 thresholds such that if the same thresholds

are crossed in both responses, the compound is considered a potential mimic. This data provides an initial assessment of the mechanism by which the receptors are activated by the

compound allowing binning of compounds that activate the receptor as the Positive Control

does (a ―mimic‖) or using a mechanism distinct from the Positive Control (―No mimic‖). For

both ER and AR, Foci Response is the primary measurement with receptor degradation (as


measured by Nuclear GFP Total Intensity) as the secondary response for ER and

translocation (measured by difference in intensity between the cytoplasm and nucleus) for AR.

In the Graphs tab, the thresholds for the secondary response are controlled by setting the Foci

Response thresholds for P1, P2, and P3. Graphing of the thresholds and flagging the response

(red, + and green, -) are performed exactly as in the Foci Response (Figures 26 and 27). Note

that receptor degradation in the ER response is a dose-dependent decrease for the Max Positive Control meaning that the thresholds are inverted on the graph as the maximum

positive response is set to 0% response and the baseline is set to 100%. For AR, translocation

is a dose-dependent increase similar to the Foci Response. The potential mimic functionality

is reported in the Conclusions tab as one of three possible outcomes (Figure 27):

N/R (No Response; green)—neither response from a compound exceeds a threshold

Mimic (red)—both responses from a compound exceed the same number of

thresholds

No Mimic (yellow)—the responses from a compound exceed different threshold

levels (i.e., P1, and P2 for foci formation and no thresholds for receptor degradation).

Figure 26. Graphs Tab of Activity templates – Visualization and comparison of response curves used in the

mimic determination. ER compares Foci Response with GFP Nucleus Intensity (A) and AR compares Foci

Response with Translocation (B).

A B


Figure 27. Using thresholds to make potential mimic determinations – Comparison of flagged responses of mimic determination in the Graphs tab are used in the Conclusions tab to assess the potential of a compound

to mimic the Positive Control.

39

Cell Imaging

For the EPP Assay, four imaging protocols are provided as a separate Dose Range and

Activity protocol for both ER and AR cells. From the ToxInsight IVT Platform software, the protocols are available in the CompartmentalAnalysis.V4 folder of Protocol Manager. The

protocols are as follows:

1) EPP_ERa_U2OS_CryoRedi_8000_20h_DoseRange_NuncEdge

2) EPP_AR_U2OS_CryoRedi_8000_24h_DoseRange_NuncEdge

3) EPP_ERa_U2OS_CryoRedi_8000_20h_Activity_NuncEdge

4) EPP_AR_U2OS_CryoRedi_8000_24h_Activity_NuncEdge

The targets and the corresponding output features from Compartmental Analysis are listed in

Tables 1 and 2. Some specific Compartmental Analysis protocol details that are required for

the EPP assay are described below.

Fixed Exposure versus Autoexposure

To compensate for plate-to-plate variation in Hoechst staining, each protocol is set to

autoexpose a single field from two Vehicle Control wells and two Max Positive Control wells

(Figure 28). If a different well is to be used for Autoexposure, you must change the ToxInsight IVT Platform protocol accordingly. If you choose Fixed Exposure, you need to

determine an appropriate exposure time for each channel for each ToxInsight IVT Platform

protocol. The saturation levels for each channel should also be monitored for each individual plate to ensure that the pixel intensities are not over- or under-saturated (refer to the

Compartmental Analysis BioApplication Guide for additional discussion on Fixed Exposure

versus Autoexposure).

Figure 28. Autoexposure - Default Wells for Dose Range templates (left) and Activity templates (right) set to

autoexpose one field from two Vehicle control wells and two Max Positive control wells

Chapter

4

40 Chapter 4 Cell Imaging

Ensuring that Wells without Cells are not Rejected

A sparse well or field, or even a field or well with no cells has meaning in toxicity assays, as one of the features being monitored is cell loss. Thus there should be no limit in the number of

sparse fields per well and sparse fields and wells should not be rejected but rather noted. The

protocols have the limit for ―Max Sparse Field For Well‖ turned off in the Create Protocol

windows. Thus for successful monitoring of cell loss, keep this limit turned off (Figure 29). The Max Fields Per Well is set to 10 to yield approximately 200 to 300 cells for Vehicle

Control wells where cell loss is minimal.

Figure 29. Counting all wells. “Max Sparse Field For Well” limit in Create Protocol window is turned off to enable successful monitoring of cell loss as a toxicity indicator.

Object Selection and Gating

The Compartmental Analysis BioApplication allows Object Selection and cell-by-cell gating

to exclude cells that are outside of selection criteria and prevent them from being analyzed.

For the Dose Range protocols, Object Selection has been relaxed for nuclei size and

fluorescence intensity to allow most nuclei to be included in analysis. Condensed nuclei may show an abnormal or high receptor response by causing Hoechst bleed-through into the GFP

channel or result in bright GFP intensity that are detected as a foci response by the algorithm.

This may be seen as a dramatic increase in receptor response in doses that are also positive for toxicity. It is suggested that if a high percentage of objects are rejected, the nuclear selection

and GFP gating features in the protocol should be adjusted to ensure that the objects are being

selected appropriately (refer to the Compartmental Analysis BioApplication Guide for

additional details on Object Selection and Gating).

Turn off the limit “Max Sparse Fields For Well”

41

Post-Imaging Analysis for EPP Prediction

The quantitative data obtained from analysis of the imaged fluorescent cells is used to make

both toxicity and receptor activity predictions. This data is imported into the proper Microsoft Excel Template, which then analyzes the data to make the response predictions.

Microsoft Excel Template for the EPP Assay

A critical component of the ToxInsight IVT Platform is the four EPP Assay Analysis Templates. One of the challenges of the new trend towards in vitro testing is the analysis and

interpretation of biological data. The EPP Assay Analysis Templates support data analysis and

toxicity and receptor response predictions for compounds with broad mechanisms of action because they monitor multiple, independent indicators within each analyzed cell. Intuitive

visualization tools are built into the templates that enable the researcher to accurately predict

drug-induced toxicity with maximum receptor activity. Outlined below are the instructions for using the EPP Assay Analysis Templates.

Guidelines

For the EPP Assay Analysis Templates to properly generate valid results, some guidelines must be followed when running plates for analysis using the template. The guidelines are as

follows:

Plate setup should be followed exactly. If a full plate of compounds has not been tested, the empty wells should be designated as blank wells or not scanned.

You should have the proper type and number of vehicle and positive controls as the template uses the vehicle control wells to normalize the data across plates.

Compound names must be added for all wells. This includes Vehicle, Positive, Sample,

and Blank wells.

The UniquePlateId for all plates must be unique.

For the Dose Range Template, only one plate can be loaded; for the Receptor Activity Template, a maximum of three plates can be loaded but data normalization and analysis is

performed on a per-plate basis.

Template Constraints

Maximum # Plates 1 for Dose Range, 3 for Receptor Activity

Maximum # Wells 96 for Dose Range, 288 for Receptor Activity

Maximum Compounds 6 sample compounds

Minimum Channels 2

Maximum Concentrations 10

Required BioApplication CompartmentalAnalysis.V4

Kinetic Data Compatible? No

Chapter

5

42 Chapter 5 Post-Imaging Analysis for EPP Prediction

Installing the Template

The EPP Assay Templates can only be populated on a computer with the Thermo Scientific AddIn for Microsoft Excel installed. Currently, this AddIn for Excel is installed on all ToxInsight instrument computers.

Please note that if you are connecting to an Oracle® database Cellomics Store, you must install the Oracle client. To install the Thermo Scientific AddIn for Excel or the Oracle client, please refer to the Thermo Scientific AddIn for Microsoft Excel User‟s Guide.

The EPP Assay templates will be installed on the ToxInsight IVT Platform computer for the

default user. In order for the template to show up in the Microsoft Excel Available Templates list, the template must be installed for each Windows user login who wishes to use the

template file.

Modifications to the Microsoft Excel worksheet can be made once the template has been saved as a new Excel worksheet (.xlsx).

Making changes to the root template files (EPP_DoseRange_ERa.xltx, EPP_DoseRange_AR.xltx, EPP_ReceptorActivity_ERa.xltx, or EPP_ReceptorActivity_AR.xltx) may result in erroneous results when using the template and AddIn to analyze results.

To install the template,

1) Copy the ThermoExcelTemplateInstall.exe file onto the local computer.

2) Login to the Windows computer as the user that you will be logged in as when using the

template.

3) Double-click the ThermoExcelTemplateInstall.exe file.

A Console window showing the progress of each template installation will appear as shown in the following figure.

4) When all template installations have completed successfully, press any key to exit the

Template installer.

The installation process copies the template into the designated local Microsoft Office Template directory for the currently logged in user. To allow multiple users on a computer to use the template, the template must be installed for each user.

Chapter 5 Post-Imaging Analysis for EPP Prediction 43

Template Workflow

The following diagram illustrates the workflow for using one of the EPP Assay Templates.

Each step in the template analysis process is detailed below.

Figure 30. EPP Assay Template Workflow

For accurate prediction, the data acquired using the EPP Assay Cartridges should be analyzed with the provided Thermo Scientific AddIn for Microsoft Excel and Excel Template for the EPP Assay.

The Thermo Scientific AddIn for Microsoft Excel and a copy of the EPP Assay Excel template

are already installed on the ToxInsight instrument computer. EPP Assay Excel templates (the

.xltx extension indicates a template file) consist of:

For ER Dose Range, select EPP_DoseRange_ERa.xltx

For ER Activity, select EPP_Activity_ERa.xltx

For AR Dose Range, select EPP_DoseRange_AR.xltx

For AR Activity, select EPP_Activity_AR.xltx

Please thoroughly review this document as well as the instructions for the Microsoft Excel

AddIn tool prior to using the EPP Assay Templates.

Loading the Template

If you try to open a new worksheet via the Quick Access Toolbar, you will not see the template screens below.

To load the template,

1) After running the assay and the images have been spooled to the correct location, open

Microsoft Excel.

2) On the Microsoft Excel Ribbon, click the File tab, and then click the New option on the

sidebar below the tab as shown in the following figure.

Run Assay

Campaign

Configure Data Source

(if needed)

Select Plate(s) to

Use in Analysis

Enter Experiment Design for each

Selected Plate

Run Template

and Save Results

Load Template and Save as Workbook


3) Click on the My Templates option under Available Templates.

4) A window showing available templates will open, click on the Thermo tab as shown

below.

5) Select one of the four templates (EPP_DoseRange_ERa.xltx, EPP_DoseRange_AR.xltx,

EPP_ReceptorActivity_ERa.xltx, or EPP_ReceptorActivity_AR.xltx) that correspond to the assay run. Click OK. The Excel template file will load. The .xltx extension indicates a

template file.


Choosing the wrong template may result in erroneous analysis and results. Verify that the biology, protocol, and the template match (i.e., AR Activity Assay run with the AR Activity protocol and analyzed with the AR Activity template).

Be sure that no other Thermo templates or workbooks are currently open when using the AddIn. The AddIn modifies all open workbooks that contain valid Thermo template definitions.

6) Before loading data, save the template as an Excel workbook by clicking the File -> Save

As… button.

****Important: Be sure that the Save As type dropdown indicates the file will be saved as an

Excel Workbook (*.xlsx). If it is not, then you may risk corrupting the template file.

7) Select an appropriate location and filename to save your soon-to-be populated Microsoft

Excel file. Click Save to save the template.

Configuring the Data Source

The AddIn can read data from a Store database or from local Plate databases in a CellData

directory. However, the data source must first be configured properly to retrieve data from the

Store database or a local directory.

If a data source had been configured at an earlier date, or if an existing Thermo Scientific

vHCS: Discovery Toolbox is installed, the AddIn will automatically default to using the last

used data source.


Configuring the Data Source for Store Database Retrieval

If connecting to a Store database to retrieve data, the HCS Applications Service must be configured correctly.

To configure the HCS Applications Service,

1) Select Cellomics Store from the Data Source drop-down in the upper left corner of the

Thermo ribbon.

The Thermo Scientific AddIn for Microsoft Excel assumes an HCS Applications Service version 3.6, which uses a default port of 54660. If the AddIn is being configured to use an HCS Applications Service version 1.5, then you must change the port number in the Advanced tab to 54550 as shown in the following figure.

2) If an HCS Applications service has been successfully configured, a list of available Store

databases will be displayed. Choose the desired Store and click Select Store. The plate

selector will be configured to query plates from the selected Store alias.

3) If an HCS Applications Service has not been successfully configured or is not connected,

the Store Options list will be empty and you will be instructed to modify the HCS

Applications Service settings in the Advanced tab. See the AddIn Installation Guide for

additional information.


Configuring the Data Source for Local Plate Data Retrieval

To search a directory for local Plate databases,

1) Select Stand Alone from the Data Source drop-down in the upper left corner of the

Thermo ribbon. The Stand Alone Configuration Window will appear.

2) Browse to the desired directory that contains plate databases (typically C:\CellData on

instrument computers) and click Connect.

The plate selector will be configured to query the Plate database files in the selected directory.


Selecting Plates

To load plates for analysis using an EPP template,

1) Click on the Launch Plate Selector icon. The Plate Selection Utility Window will open

as shown below.

Upon opening, the plates from the selected data source will be automatically filtered by

the BioApplication specified (in the case of the EPP templates, the BioApplication is

CompartmentalAnalysis.V4).

2) To add additional criteria, click on the criteria bar on the left, enter a value for the selected

criteria to match, and click the green arrow. The plates will then be filtered by the

specified criteria.


3) The template supports the analysis of up to three plates simultaneously (for Activity

Templates), with one plate required as the minimum for analysis. Dose Range Template

can only support analysis of one plate at a time.

To select plates for analysis, click on the green plus icon to the left of the desired plate.

The plate will be moved to the Selected Plates grid.

To remove a plate from the Selected Plates grid, click on the red ‗ – ‘ icon to the left of

the plate.

To clear all plates in the Selected Plates grid, click the Clear All button above the

Selected Plates grid.

4) After all plates have been selected, click the Import Data button to load the raw data from

the plates into the template. The Plate Selection window will close.

Clicking Close before importing the data will result in the clearing of the selected plates list.

After importing the plates, the experiment design or plate layout of each plate will need to be specified in order to run the template.

Entering Experimental Design Information

To enter experimental design information,

1) After plates have been selected using the Launch Plate Selector window, click the

Launch Experiment Design icon to load the Experiment Design window.


2) Select the plate(s) to annotate and click the Annotate button to display the annotations in

the plate grid below.

Icons to the left of the plate name indicate one of three possible plate annotation statuses:

? – Indicates that no annotations have been assigned to the plate

! – Indicates that the plate is partially annotated. Additional annotations are needed

to run the analysis

– Indicates the plate annotation has been completed and that the plate is ready for

analysis using the EPP templates.

Multiple plates can be annotated at one time if the plate status is (?) and no annotations have been assigned to the plate. This is typically useful in the case of replicate plates.

If plates have been fully or partially annotated, then they will not be available for multi-selection.

If you select three plates for analysis and all plates have the same well type layout and concentration ranges, you can select three plates and annotate the well types and concentrations for the plates.


3) Select the wells for annotation. Wells are selected for annotation by directly clicking on

them (selected wells are highlighted in blue). Clicking on highlighted wells deselects

them. Clicking on column and row headers selects/deselects individual columns/rows.

Clicking on the upper-left box will select/deselect the whole plate.

4) Once wells have been selected, specify experimental design information for each plate.

For the EPP assay analysis templates, each well must have the following annotations

assigned:

Well Type

Compound

Concentration (use the same units for each well)

Use the same units for concentrations for each well (i.e., nM). Inconsistencies in units during annotations (labeling some wells with nM and others with mM) will cause erroneous analysis and results.

The Well types available for the Dose Range templates are: Blank, Vehicle, Max

Positive, and Sample. For Receptor Activity templates, the Well type choices are:

Blank, Vehicle, Max Positive, Positive, Weak Response, and Sample.

The Vehicle and Positive controls are used to normalize the data for each plate. Therefore, it is highly suggested to use the suggested plate map for all templates.

5) After annotating one or more plates, click the Apply button. Then, select a single plate to

add compounds to and click Annotate.


6) When ready to select another plate from the grid, save the current annotation, highlight the

desired plate to annotate, click the Annotate button, and follow steps 1-5 above. Once

annotations for a plate have been added, click the Apply button to save any changes.

Failure to do so may result in losing annotation designations.

7) To turn annotations on or off in the annotation grid, check or uncheck the Well Type,

Compound, or Concentration boxes on the toolbar. Doing so simply shows or hides the

annotation(s) on the grid.

8) When all plates have been annotated and have complete statuses, click the Finish button to

populate the template.

Once the template has been run, data will be populated and the analysis will be available for

viewing.

It is important to complete annotations for all selected plates, and complete running the template. The same units for concentrations for each well (i.e., nM) must be used per plate in order to prevent erroneous analysis and results.

In order to complete the experiment design and run the template, all wells must be annotated. If wells were not scanned or are not of interest for the analysis, they must be annotated as Blank. Compound names and Concentrations must be added for all wells. Blank wells can be annotated with “Blank” in the Compound name and “0” as the concentration.

If 1% DMSO is used as the Vehicle Control, „%‟ cannot be selected for the concentration‟s units. However, since this concentration value (nor units) is used in data analysis, it is suggested to annotate it as 0.0001 using the unit‟s designation that is consistent for the plate. Using 0 as a value will cause the template to error since 0 cannot be used for logarithmic values.

Compound names used for annotations are retained only for the active session. At the end of the session, when you close the template, compounds names are deleted from memory. So for the next analysis session, you will have to re-enter the compound names again for the new analysis. Compound names can be cut and pasted from other sources using text lists or semi-colon delimited text.

Running the Template

After plates have been selected and annotated, the template can be run. When you click the

Finish button in the Experiment Design window, the AddIn for Microsoft Excel runs the

template by automatically importing raw data from the configured data source and associating the raw data with the user input experiment design data into the Rawdata worksheet (see below

for details). The AddIn automatically populates the template and performs the pre-determined

calculations, writing lists of data into the template as specified by the Template Definition.

List data are derived by the AddIn and template following annotation to match the raw data to

the correct annotation and then to pre-defined calculations (i.e., matching up data from Vehicle Control wells with the formulas that determine the Vehicle Control mean). Once the raw data

and list data is written into the Microsoft Excel spreadsheet, the analysis is completed through

formulas, graphs, and formatting pre-populated in the template. Final analysis requires user input to define thresholds and compound selection (for both graphing and data visualization).

All yellow cells within the template are user-interactive and can either be changed via a pull


down menu or require user input. Several tabs (Basal Response, Normalized Response, QC,

Graphs, and Conclusions) have pre-defined print areas. See the Worksheets section below for

information about individual template worksheets.

Worksheets

Each EPP template contains multiple worksheets that are utilized for distinct steps in data

analysis. Several worksheets are repositories for imported or normalized data sets and do not require user input. Other worksheets require or allow for user adjustments. There are seven

pre-defined worksheets in the template. A description of each worksheet is provided below.

Worksheet 1: Rawdata

Deleting data or formulas from the Rawdata worksheet will directly result in changes to the end result and may corrupt any of the EPP Assay analysis templates.

The data from the Rawdata worksheet may be sorted without affecting the end result.

This worksheet contains the raw data imported from the Cellomics Store database or

standalone database(s) that you selected in the Plate Selector window and input in the

Experiment Design window. The Rawdata worksheet includes the following information:

Plate Location (Store alias or local file)

UniquePlateID (Unique Plate Identifier)

Row (1-based)

Column (1-based)

Compound

Well Type

Concentration

Concentration Units

Replicate

Object Count

Nucleus Intensity (Dose Range templates only)

Nucleus Size (Dose Range templates only)

Foci Intensity

Foci Area

Foci Count

GFP Nucleus Intensity (ER Dose Range and Activity templates only)

Translocation (AR Dose Range and Activity templates only)


If a well was determined invalid or sparse by the software (as defined by the Assay Protocol),

the well may not be listed in the Rawdata worksheet. If a well contains only some of the required features, the well will still be listed, but may not have values for all of the required

features.

Figure 31. RawData Worksheet

Worksheet 2: Basal Response

This worksheet calculates the average basal response for both the Vehicle and Max Positive

Controls on each individual plate and monitors data quality used for Plate Acceptance criteria.

The Basal Response values will be used to calculate the normalized response for each well on the plate. This tab uses data from the Rawdata tab to determine the basal levels of the control

values.

Basal Level Determination

For nuclear features, the basal levels are defined as the average of the Vehicle Control

wells.

For GFP features, the average of the Vehicle Control wells is defined as the baseline

response. The basal response is calculated by subtracting the baseline from the average Max Positive Control value (MPCmean – VCmean). The determination of

receptor degradation in ER templates (GFP Nuclear Intensity) is calculated differently for GFP basal responses (as it is a dose-dependent decrease) by subtracting

the mean Max Positive Control average (baseline) from the Vehicle Control average

(VCmean – MPCmean).

If multiple plates are imported into the Activity templates, basal responses are determined individually on a per plate basis and the controls (Vehicle, Max Positive, Weak Response, and Positive Controls) from each plate are not averaged together.


Figure 32. Dose Range Basal Response Worksheet (top) and Activity Basal Response Worksheet (bottom). Mean basal response is calculated for each plate that is imported. This number is used to normalize the response for each well.

Plate Acceptance Criteria

Additionally, the Basal Response tab monitors plate data quality assessing plate health by

utilizing plate acceptance criteria that ensures variation on the plate is below defined cut-offs. Data quality is assessed through the determination of Coefficient of Variation (CV)

derived from the raw data of each type of control wells and comparison of Background

Response and Fold Induction. See Assessing Plate Data Quality and Reviewing Plate

Acceptance Criteria (Chapter 3, Step 2) for a detailed description of the calculations. If multiple plates are imported into the Activity templates, data quality for each plate will be

assessed and monitored individually.

Recommendation: We recommend that you verify the data quality for each plate (all

outputs in the Quality column should report ―Pass‖) and investigate any ―Verify‖ values through the QC tab or inspection of the images or data in the iView software. A print area

is set for the Data Quality Table for tracking of Plate Acceptance criteria.


Figure 33. Dose Range Basal Response Data Quality table


Worksheet 3: Normalized Data

Deleting data or formulas from the Normalized Data worksheet will directly result in changes to the end result and may corrupt the EPP Assay analysis templates.

The data from the NormalizedData worksheet may be sorted without affecting the end result.

The Normalized Data tab uses the Basal Response data to normalize the data from the

Rawdata tab. Data is normalized on a per plate basis and viewed in list form.

For nuclei features, normalization is performed by dividing the Rawdata by the

Vehicle Control basal response.

For GFP features, normalization is performed by first subtracting the Vehicle Control

basal response from the Rawdata and then dividing by the Max Positive Control basal

response. The determination of receptor degradation in ER templates (GFP Nuclear Intensity) is calculated differently for GFP normalized data as it is a dose-dependent

decrease response by subtracting the mean Max Positive Control basal response from

the Rawdata and then dividing by the Vehicle Control basal response.

Figure 34. Normalized Data Worksheet

Note that all cells shaded in grey contain formulas. Deleting or modifying these formulas may corrupt the results.


Worksheet 4: Normalized Response

This tab averages together the replicates on the plate to provide a single normalized response for each dose. Also note that the values derived for each feature are from the user-defined thresholds

and are displayed on this tab. This worksheet contains the following:

The average normalized responses across replicates for each feature, compound, and

concentration

A table used to calculate threshold levels

Listing of all compounds

A print area is set for the Control Statistics Table for tracking of the standard deviation of each

feature and the derived threshold levels.

Figure 35. NormalizedResponse Worksheet

Control statistics used for calculating thresholds

Calculated average normalized responses for each compound and concentration combination for all features

Compounds


Worksheet 5: QC

This sheet contains options to view Compound, Concentration, Units, Replicates, or WellType, and a heat map for each plate and feature for the purpose of QC. The heat map is based off of the

NormalizedData tab.

The quality of the cells within a plate can be monitored in the QC worksheet by examining the

control wells. Good quality cells will have minimal variation between control wells (i.e., Vehicle-

treated) for each measured feature.

Recommendation: This tab can be used to verify the plate annotation and to visualize normalized

data. This may be helpful to identify outlier wells that may result in poor data quality. A print area is

set to include both the Plate Annotation and Select Feature to View values.

Figure 36. QC Worksheet

There are also several pre-defined named ranges, formulas, and populated cells in the template. Removing any of these may affect the integrity of the template results.

Select Unique Plate ID and Annotation to show for the selected plate (e.g., Compound, Concentration, WellType, or Replicate)

Select feature to view for the selected plate. The normalized values are displayed.


Worksheet 6: Graphs

This tab contains options to display graphs for the compounds and features (concentration on the x-axis, normalized responses/levels on the y-axis) as well as the calculated response. The

calculated responses are flagged red (+) if any dose from a compound is outside of the defined

thresholds and is green (-) if all doses from a compound are within the thresholds. This tab

also contains the tunable thresholds used to change the output responses. The data used to

generate the graphs and flagged responses is shown below the graphs in a table format.

Up to 8 compound/replicate choices (or the mean response for a compound) may be selected

to view on the dose vs. feature and dose vs. multi parameter index graphs.

The graphs show the response for the selected compounds and replicates as well as the

calculated toxicity thresholds (dashed lines) based on the user-defined inputs (See Chapter 4 for details on setting thresholds).

This tab requires user input to populate the Compounds to Graph column Note that the table and graphs will be empty until compounds are selected in the Compounds to Graph column).

Besides compound selection, the Replicate (Rep) column and thresholds can also be adjusted

and will result in changes in the flagged response table and graphs (adjusting the thresholds or

the visualization of compounds/replicates does not alter the normalized data). A print area is

set to include the flagged response table, threshold table, and graphs.

Figure 37. ER Dose Range Graphs Worksheet. Graphs and features will differ on other templates.

Select Compound and Replicate or Mean from the dropdown lists in this table. The results for the selected compound mean or replicate is shown to the right.

Feature graphs for the selected compounds

Multi-parameter indices based on the concentration and mean response for the selected compound


Worksheet 7: Conclusion

This tab provides detailed analysis of the response results, graphical visualization of the primary results, and the experiment information and data quality summary. This tab also provides final analysis of the

data. Data used to generate the final analysis is shown to the right of the graphs.

For the Dose Range templates, the Conclusions tab suggests a starting dose response for the

subsequent Receptor Activity workflow basic on Excel logic statements. See Figure 8 for more

information.

For ER , the starting concentration for the Activity Assay is the maximum non-toxic dose and minimum dose + 1 that gives maximum foci response above the user-defined

threshold.

For AR, the staring concentration for the Activity Assay is the same as ER unless there is

no foci response and then it is the minimum non-toxic dose + 1 that gives the maximum

translocation response.

For the Activity templates, the Conclusions tab provides detailed response analysis via comparison

with the Max Positive Control and/or Positive Control showing:

Threshold-based flagging of receptor activity

Potency information derived from linear regression analysis

Magnitude of the receptor response

Determination of the potential mimic function

An experiment info table is available for user input of pertinent information. A print area is set to

include the flagged response table, experiment info table, plate acceptance summary, and graph.

In addition, you can input experiment information (such as the experiment name, plate identification

numbers, and user info) and use several pull down menus (designated in yellow) to select the

compounds or results that will be displayed.

Recommendation: This tab requires user input to populate the Plate ID and compounds column. The

template specific inputs can also be adjusted and may result in changes in the flagged response

table and graphs. Adjusting these values may alter the final analysis outputs; please be sure that

the desired settings are selected. For example, changing between Toxicity Index and Viability in the

Dose Range graph may result in different suggested starting doses.


Figure 38. Conclusion Worksheet – Dose Range

Figure 39. Conclusion Worksheet – Activity

Selected Compound for Visualization and suggested starting concentration

Graph incorporating choice of toxicity and foci response

Information entered by user for experiment tracking

Graph incorporating compounds with threshold markers for each

Information entered by user for experiment tracking

Concentrations calculated from thresholds

Plate validation summary

Selected Plate and Compound for Visualization

Plate validation summaries


Suggested User Template Worksheet Workflow

After running the Thermo Scientific Microsoft Excel Add-In, relevant data is imported, organized, and analyzed. The template automatically populates each worksheet but requires

user adjustments and monitoring to adequately report receptor responses. Comments (seen as

red marks in the corners of cells in the Microsoft Excel worksheets) have been added to the

templates providing easily accessible descriptions or information for designated worksheet

cells. Comments can be viewed by positioning the cursor over the cell.

Figure 40. Comments – position the cursor over the red triangles to view comments

For each template, there are three main areas from all the tabs that may require user inspection

and/or adjustments:

1) Basal Response: This worksheet provides the determination of the control data

quality from the plate(s). If the data passes all of the listed criteria, no further action is required. If any items are flagged or fail to meet set acceptance criteria, an

investigation into the failure must be performed and the failure resolved prior to data

from the plate being used for further analysis. This could be through inspection of the

imported data to ensure no errors during import, viewing the data on the QC tab, or could require additional inspection of the original data or images in the iView

software.

A summary of the data quality is also reported on the Conclusions tab.

2) Graphs: This tab allows you to graphically visualize the data and determine

thresholds for each feature. You can also inspect dose responses for each replicate

individually compared to the mean to further examine any variability within the assay. Once acceptable thresholds have been established, constant adjustment of levels for

additional plates should not be necessary, but monitoring control responses for

variability and expected results is recommended.

3) Conclusion: This worksheet requires some data input (template name, experiment

number, etc) and provides the ability to define the final outputs.

The other tabs are freely accessible to allow transparency in all data analysis. Be careful not to change any data outside of those boxes highlighted in yellow as it may alter the original data or delete a link that could drastically impact the final result.

65

Sample Preparation Using the EPP ERα and AR

Assays

Important Information

Please refer to the Instruction Booklet entitled: Consumer Health and Product Safety – Thermo Scientific ToxInsight Endocrine Profiler Panel Cartridge: Estrogen Receptor alpha and Androgen Receptor Assays for complete instructions on cell and sample preparation, as well as both assay procedures.

The Thermo Scientific EPP Cartridges can be purchased directly through your local Thermo Fisher

Scientific sales representative. Order numbers for each kit are the following:

Number Description

R04-1001-ERa ToxInsight Estrogen Receptor α Cartridge, materials for 7 96-wells (6 compounds)

R04-1001-AR ToxInsight Androgen Receptor Cartridge, materials for 7 96-wells (6 compounds)

STORAGE: Cartridge(s) will arrive in 2 separate packages. Package #1 will contain microplates and plate seals, media, bottles for the different media types, buffers,

and Hoechst. Store all buffers, media, and dyes at 4°C protected from light. The remaining items can be stored at room temperature.

Package #2 will contain the cells, serum, media supplements, and labels for the different media types. Store

the cells in vapor phase of liquid nitrogen and media supplements at -20°C.

Allow the media, supplements, serum, buffer, and dyes to warm to room temperature before use. See the

Solution Preparation section in the Instruction Booklet for storage and stability of prepared solutions.

If you are testing where regulation requires you to document proper routine instrumentation calibration, please refer to the Thermo Scientific ToxInsight IVT Platform Illumination Correction and System

Calibration Guide (LC063238) and Thermo Scientific ToxInsight IVT Platform Illumination

Correction and System Calibration Results Sheets (LC063239) for additional information. We suggest

that you run these tests PRIOR to setting up the biological protocols. If you would like to perform routine system calibrations, we suggest that you order the following:

K10-0099-1 Thermo Scientific OptiTracker Optical Performance Monitoring Kit

Chapter

6

66 Chapter 6 Sample Preparation Using the EPP ERα and AR Assays

Additional Material Required

Dimethyl Sulfoxide (DMSO; suggested vendor – Sigma #D2438, CAS 76-68-5)

16% Paraformaldehyde (suggested vendor – EMS #15710, CAS 50-00-0)

Positive and weak positive compounds (see Table below for recommended compounds and vendors). Controls should be adhered for proper Dose Range and Activity Assays (setup as

recommended plate layouts in Figures 5and 6 of the Assay Guide)

You will need a Schedule 3 license from the DEA in order to use DHT (or other Schedule 3 compounds within the United States). Please see http://www.deadiversion.usdoj.gov/drugreg/reg_apps/onlineforms_new.htm for more information (note that each state has different requirements). For customers outside the United States, please consult proper regulatory guidelines for handling Schedule 3 compounds.

Sterile distilled H2O for moat in plates (suggested vendor - HyClone # SH30529.02)

Distilled deionized H2O for buffer dilutions

Liquid Nitrogen Dewar (or equivalent) for storing cells prior to use

A general assay workflow is given in the figure below. Note that the Dose Range Assay must be

done prior to the Activity Assay.

Compound Vendor Catalog # CAS# Type

β-estradiol Sigma E8875 50-28-2 ERα Positive Control

Bisphenol A Sigma 239658 80-05-7 ERα Weak Response Control

DHT Sigma A8380 521-18-6 AR positive control

Progesterone Sigma P0130 57-83-0 AR Weak Response Control

Dose Range Assay

Receptor Activity Assay

Determine

Starting Dose

Plate Cells Treat 1 plate

(6 compounds)

Plate Cells Treat 6 plates (2 compounds,

duplicate plates)

Fix and Stain

Fix and Stain

Scan and assess Toxicity vs. Receptor

Response

Scan and analyze Receptor Response

http://www.deadiversion.usdoj.gov/drugreg/reg_apps/onlineforms_new.htm

US Office

Thermo Fisher ScientificLife Science ResearchBiosciences Division100 Technology DrivePittsburghPA 15219USA

Tel: 1.412.770.2200Fax: 1.412.770.2450Email: [email protected]: www.thermoscientific.com/cellomics

European Office

Thermo Fisher ScientificIndustriezone IIIIndustrielaan 279320 ErembodegemBelgium

Tel: +32 (0)53 85 71 82Fax: +32 (0)53 85 72 28Email: [email protected]: www.thermoscientific.com/cellomics

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