Guided Analytics for FLIPR fluorescence processing in biotech R&D

Tod Flak, Ph.D.Head, Lab Informatics & Automation

tod.flak.tf@axxam.com

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