guided analytics for flipr fluorescence processing in biotech r&d tod flak, ph.d. head, lab...
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Guided Analytics for FLIPR fluorescence processing in biotech R&D
Tod Flak, Ph.D.Head, Lab Informatics & Automation
Axxam srlSan Raffaele Biomedical Science Park
Milan, Italy
About Axxam
AXXAM is located at the San Raffaele Science Park, and has more than 1,400 square meters of fully equipped laboratories and office space
November 2001 entered a five year research agreement (worth € 30 million) with Bayer AG
AXXAM srl was founded May 2001 in Milan
Our team consists of 50 people
Axxam: From Genes to Leads
Business model Industrial collaborations and services Internal R&D for technologies and know-how / IP position Currently several pharma/biotech partners
ActivitiesTarget identification and validation Development of screening assays for lead
identification (HTS, uHTS, etc...)Screening and lead profiling
Industrial areasPharmaceuticalDiagnosticAgro-chemical
AXXAM: The Assay Factory
Over 20 clean benches, 30 cell incubators
Multiple Pipetting robots: Matrix and CyBio workstations (96/384/1536 well formats); Packard Multiprobes
2 FACS stations
6 Luminescence readers (3x 96 and 3x 384 well format)
3 Fluorescence readers (1x 96, 1x 384 and 1x 1536)
FLIPR 384 well workstation• Assay development and validation• Screening services
More than 100 screening assays are already available
FLIPR based Assays for HTS
• FLIPR® (Fluorometric Imaging Plate Reader) system from Molecular Devices is an industry-standard platform
• CCD-based plate reader with integrated plate handling and pipetting (384 channel)
• Axxam has configured FLIPR assays for multiple gene classes:
•GPCRs coupled to Gq
•GPCRs artificially coupled to Gq
•Ligand gated ion channels
•Voltage gated Ca2+ and K+ channels
•Electrogenic transporters
•Ion exchangers
All assays are optimized for the use in 384 well format
• Instrument used for internal development and also high-throughput screening for partners
Requirements for FLIPR data analysis
clone 13.12.3 48hr
10 -1.50 10 -1.25 10 -1.00 10 -0.75 10 -0.50 10 -0.25 10 0.000
100000
200000
3000001500 c/w1750 c/w2000 c/w
K+ (M)
RL
U (
20 s
ec i
nte
gra
l)
• Specific import of FLIPR data format
• Curve-fitting
• Adapt easily to research & development phase :: single plates, never the same plate layout twice!
• Easy to use
• Customizable
Spotfire FLIPR Guide
• Walks users through analysis steps
• Reorganization and summarization of data
• Visualize data at all steps
• Mark bad data
• Integrated Curve fitting – using Excel (no need to purchase specialized curve-fitting software)
• Open code -- easily customizable
• All the other benefits of Spotfire
Demo of basic FLIPR Guide
Customization of FLIPR Guide
If only used in processing HTS data, no customization needed– Plate layout fixed– Typically stimulate with known agonist, experimental
compounds are potential inhibitors
In assay development, need to change things such as:– Plate layouts– Filenames– Variable of interest – optimize agonist concentrations
(agonist alone), incubation times, etc.
Plate Layout Tool
• Users can define layout on-the-fly
• Integrated into Spotfire guide workflow
• Built in Excel
ATP-sensitive Potassium Channel• KATP channels are formed as a complex of :
sulfonylurea receptor (SURx):
SUR1 (pancreas)SUR2A (heart)SUR2B (smooth muscle)
& inward rectifier (Kir6.x) subunit:
Kir6.1Kir6.2
• Family of weak inward rectifiers
• Distinguishing feature: intracellular ATP inhibits the channel, increase in intracellular MgADP activates the channel
C
NSUR1 Kir6.2
K+
OUT
IN
ATP
glibenclamide
Openers MgADP
++
Complex Voltage-sensitive experiment
-5000
-4000
-3000
-2000
-1000
0
1000
2000
3000
4000
5000
00 1 2 3 4 5 6 7 8 9 10Time(minutes)
Multiple Well Overlay
Flu
ore
scen
ce C
han
ge
(Co
un
ts)
100 uM Diazoxide (induces K+ outflow)
Glibenclamide
(blocks channel, restores intracellular potential)
High Glibenclamide
Low Glibenclamide
• Voltage sensitive dye
• Monitoring depolarization and restoration by inhibitor
-3000
-2000
-1000
0
1000
2000
3000
4000
5000
6000
7000
00 1 2 3 4 5 6 7 8 9 10Time(minutes)
Multiple Well Overlay
Flu
ore
scen
ce C
han
ge
(Co
un
ts)
Complex Voltage-sensitive experiment
100 uM Diazoxide (induces K+ outflow)
Glibenclamide
(blocks channel, restores intracellular potential) High Glibenclamide
Low Glibenclamide
• Voltage sensitive dye
• Monitoring depolarization and restoration by inhibitor
Voltage-sensitive channel assay testingFirst injection: 100uM diazoxide
Second injection: glibenclamide 0.01nM -- dose curve 30uM
Summary
• For Axxam, Spotfire provided a good solution for our needs in analysis of FLIPR data
• Customization and extensibility of the tool are crucial
• We added options for – Quickly defining plate layouts– Non-standard experimental design processing
• In future we will add – More processing options– Mechanism to retrieve data using LIMS database links– Mechanism to save results into LIMS database