18-1175-31 AA

theanswer is

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INCell Analyzer 3000

high-throughput sub-cellular imaging system

wwwIf your CD is missing, please request your copy

(quoting reference code 18-1177-12) on:

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Minimum system requirements: WindowsTM

You will require a minimum of Windows 95 or later,16 bit color 800 × 600 pixels

and double speed CD Rom

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IN Cell Analyzer

3000is a purpose built sub-cellular screening system. Combining

sophisticated scanning and optical technologies with

high-throughput capacity, the system provides advanced

multiplexing capabilities and real time, cell-by-cell image

analysis routines for live- and fixed-cell assays.

Designed to run continuously: a 950 000-compound library can be screened andanalyzed in 6 weeks

Precision optics and lasers: generate high-content screening images from assayswith sub-micron resolution

Dedicated image analysis modules: perform rapid imaging and quantitativeanalysis of sub-cellular components in a range of applications

High-speed throughput and analysis: scans 30 000 standard images in 16 hours

Straightforward set up: scans standard 96- and 384-well microplates

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applications

technology

The IN Cell Analyzer 3000 enables high-contentcellular screening of live- and fixed-cell assays in thefollowing areas:

Fig 1. IN CellAnalyzer 3000enables high-contentsub-cellular imagingand analysis in ahigh-throughputscreeningenvironment.

IN Cell Analyzer 3000 achieves high-resolution imagingand analysis through a combination of optimizedconfocal optical pathways, dynamic autofocus,and cooled digital cameras. The environment of thesample chamber can be set to maintain temperature,humidity, and CO2 levels at optimal cell cultureconditions for live-cell assays. The graphical userinterface combines flexible and intuitive software andanalysis modules capable of rapid scan rates andquantitative data analysis.

Lead compound discovery

Target validation

Functional genomics and proteomics

Metabolism and toxicology

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Confocalline-scanningbeamand large fieldof viewThe configuration of the scanning beam (0.6 x 750 µm) (Fig 2)and the very short sample exposure time allows live cells to bescanned at high resolution in less than 3 s without adverselyeffecting their physiology or photo-bleaching fluorescentmolecules. This means that cells can be scanned repeatedly duringkinetic studies or re-scanned if necessary in static studies.

Confocal optics collect images from a defined focal plane, whichreduces sample background, increases sensitivity, and caneliminate liquid handling steps in assay processing, facilitating theperformance of homogeneous assays.

A large field of view (750 x 750 µm) ensures that a high cellcount can be achieved in every scan (up to 500 cells per field at40× magnification). This means statistically significant data arerapidly acquired from cell populations.

Fig 2. Confocal line scanning with parallel pixel illumination and detectionenables rapid imaging without probe or sample degradation. The arrowdescribes the passage of the scanning beam over the cells and the yellowline defines scanning laser length and width.

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DynamicautofocusIN Cell Analyzer 3000 incorporates an infrared laserline that automatically detects the interface betweenthe liquid in the well and the bottom of that well(Fig 3). The system then focuses the objective lens oncells that are in a user-defined focal plane above thisinterface. As a scan proceeds, tracking autofocuscontinuously adjusts the focus of the objective lens onthis defined focal plane, ensuring that all scanned cellsare in focus. This process reproducibly generates imagesthat are optimal for high-content cellular analyses.

Fig 3. Dynamic, continual autofocus enables very rapid plate scans and cellular screens by ensuring imaged samples are constantly in focus.

Continuous, infrared laser autofocus ensures rapidprogression from well to well because the sample isalways in focus.

The system automatically adjusts the focal plane toa user-defined offset level that generates optimalimages for quantitative analysis.

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Multiplexed, simultaneous

-colorimaging

Multiplexing facilitates the simultaneous investigationof more than one event during an assay (Fig 4). IN CellAnalyzer 3000 is the only high-throughput screeningplatform to incorporate three high-speed, cooled CCDcameras for the simultaneous imaging of red, blue, andgreen emissions from samples that have been triplelaser line-scanned.

Analyzes complex multi-parametric cellular assaysby excitation at three different wavelengths.

Generates three-color images enabling sub-populationanalyses on data generated from individual cells, ona cell-by-cell basis.

To determine the effect of cell cycle position onligand binding a GFP-based cell cycle phase marker

Fig 4. Multiplexed analysis of ligand binding and cell cycle status using a GFP-based cell cycle phase marker.

3(CCPM) was multiplexed with fluorescently labelledcholera toxin (CTX). Transition of the CCPM throughthe G2 and M phases of the cell cycle was thenmonitored (Fig 4).

A U-2 OS cell line expressing the EGFP G2/M CCPMwas incubated for 15 min at 0 °C with 0.5 ug/ml Alexa647 CTX subunit B conjugate; both in the absence(Fig 4a) and in the presence of 50 ug/ml unlabelledCTX subunit B (Fig 4b). Cells were then washed toremove unbound CTX and their nuclei stained withHoechst. Fluorescently labelled cells were then imagedon an IN Cell Analyzer 3000. Figure 4 shows binding offluorescent CTX is restricted to cells with low CCPMexpression (i.e. cells in G1).

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Please visitour Web site at:

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incellanalyzeror contact your local sales office

for the latest information.

IN Cell Analyzer 3000 incorporates a sample chamberin which the internal environment can be controlled at37 °C, 70% humidity, and 5% CO2 enabling live-cellassays to be performed in addition to fixed-cell assays.Live-cell assays yield more accurate information abouta compound's true effect within the living cell asopposed to information from assays that rely onenzymatic or nucleic acid binding data derived from invitro assay systems.

Full environmental control maintains optimal cellculture conditions for the performance of timedependent kinetic assays and extended analyticalassays on living cells.

Two pumps in the sample chamber can beprogrammed to rapidly administer agonist orantagonist compounds to cells through'non-contact' dispenser tips in volumes of between10 and 50 µl, in 10 µl increments.

Sample chamberwithenvironmentalcontrol

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Choice of application is achieved through versatileanalysis modules designed for the study of biologicallyrelevant applications in lead selection, target validation,and cellular toxicology studies, such as:

Nuclear translocation

Receptor internalization

Mitochondrial translocation

Protein expression

Apoptosis

Ligand binding

Reporter gene expression

Cell cycle analysis

Neurite extension

Cell morphology

Flexible software performs quantitative image analysisroutines at the individual cell level and in real time,during image acquisition cycles. Well images can beselectively saved via user-defined scripting options.

The current range of Image analysis modules is shownin Table 1.

Imageanalysismodules

Table 1. Image Analysis modules for IN Cell Analyzer 3000.

Module Measures Examples of use

Object Intensity Intensity changes, rapid kinetics G1ST, viability, toxicity, viral binding, calcium flux

Nuclear Trafficking Translocation: cytoplasm, nucleus NFκB, STATs, SMADs, p53, GR, Erk, MAPKAP-k2, NFAT

Plasma Membrane Trafficking Translocation: cytoplasm, cell surface PLCδ, Cofilin

Plasma Membrane Spot Subtle changes at cell surface Rac, AKT

Granularity Discrete granules: transport vesicles, Receptor internalization, nuclear condensation,micronuclei, mitochondria cytochrome C trafficking

Cell Cycle Trafficking Distinguishes cells in prophase, G2/M cell cycle phase markers assay employing a(Static) mitosis, G0/G1/S, and G2 reporter protein comprised of EGFP fused to a

fragment of cyclinB1

Morphology Flexible cell shape recognition Identification of apoptotic and necroticcells in screening assays

Granularity Update Variable grain size recognition, CypHer based receptor internalization(CypHer) automated analysis parameter

optimization, improvedbackground sampling method

Apoptosis-Nuclear Simultaneous quantitation of Various apoptotic marker proteinsTrafficking and Granularity nuclear trafficking and/or nuclear

orphology and granularity

Neurite Extension Recognizes and quantitates Retinoic acid-induced changes in PC12neurite extensions and cell bodies Neuro-2a cells

Image analysis modules facilitate high-speed cell-by-cell image analysis, online, during each scan. Run set upand execution are performed through an intuitivegraphical user interface that has fully adjustable, user-defined parameters that ensure optimal, reproducible,assay analyses.

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Examplesofanalysismodulefunction

Granularity

Nuclear Trafficking

Quality metricsfor HCS data

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GranularityAnalysisModule

Fig 6. Image of living cells expressing a fluorescently tagged endosomalreporter protein. (A) Endosomes within U2OS (human osteosarcoma) cellsare decorated with enhanced green fluorescent protein (EGFP) fused totandem FYVE domains. Location of the reporter is dependent on activity ofa phosphatidlyinositol 3-kinase, and the cell line can be used to screen forinhibitors of this class of enzyme. (B) After 30 min treatment of cells with aPI3-kinase inhibitor (100 nM Wortmannin), the EGFP-2xFYVE dissociatesfrom the endosomes and distributes diffusely throughout the cellcytoplasm. Green = endosomes (EGFP-2xFYVE), Blue = nuclei (Hoechstnuclear dye).

A B

Fig 5. Analysis of TRHR-1 internalization using the Granularity AnalysisModule. CHOK1 cells expressing VSV-G epitope-tagged thyrotropin releasinghormone receptor-1 (TRHR-1). Cells were pre-incubated with 5-mg/mlCypHer5-labelled anti-VSV-G antibody and then incubated for 30 min at37 °C with (A) no TRH or (B) 10-mM TRH. Bitmaps overlaid on the imagesdemonstrate how the Granularity Analysis Module analyzes CypHer5internalization. The analysis routine identifies a measurement region for eachcell (large boxes), and then searches within each measurement region forfluorescent grains of a user-specified size. No CypHer5 is detected in unstim-ulated cells (A). After agonist-induced receptor internalization (B),CypHer5 is detected in large perinuclear recycling endosomes (smaller boxes).

A B

A variety of biological stimuli result in the associationor dissociation of proteins or other molecules withgrain-like objects within cells. When such proteins ormolecules are tagged with an appropriate fluorescentor fluorogenic molecule, these granular objects can beimaged on the IN Cell Analyzer 3000.

Changes in the granular appearance of a cell can beassociated with key processes, such as endocytosis,receptor activation, receptor down-regulation,secretion, and activation or deactivation of intracellularenzymes. The Granularity Analysis Module provides amethod to identify, count, and analyze grain-likestructures within cells (Fig 5).

The system software generates cell-by-cell andpopulation-averaged results for each well to provideyou with complete assay information. The flexibilityof the Granularity Analysis Module enablesquantitative analysis of a range of assays that involvechanges in granular fluorescence, dissociation ofreporter molecules from cytoplasmic endosomes(Fig 6), and internalization of plasma membranereceptors (Fig 7).

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Fig 7. Graphical user interface of Granularity Analysis Module showing (clockwise from top left hand corner (A) Plate map. Yellow wells indicate cells thatcontain granules and green wells indicate absence of granules. (B) Image with bitmap defining cells present in well 'A1' and the vesicles contained within them.The degree of granularity is quantitated and recorded individually for each cell in the image. (C) Population scatter plot in which the points indicatepopulation-averaged granularity measurement for each well (in this case, one image per well). Yellow, prior to addition of agonist and green after addition ofagonist. (D) Population Summary table containing detailed, population-averaged granularity measurements for each well. Individual cell data are also recorded,but not shown here.

A B

C

D

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Fig 8. 96-well live-cell EGFP-SMAD2 assay. CHO-hiR stable cell line expressingEGFP-SMAD2 fusion protein was stimulated for 60 min in (A) the absenceor (B) presence of 3 ng/ml transforming growth factor-β1 (TGF-β1).(B) Following activation with TGF-β1, the EGFP-tagged transcription factortranslocated from the cytoplasm to the nucleus. Hoechst 33342 nuclearstain also shown. Images shown are 0.25 of the actual image size acquiredby the IN Cell Analyzer 3000. Error = +/-SD, n = 48 replicates per data point.

Fig 9. Two quality control inhibition curves obtained on consecutive daysof a screen using the GFP-MAPKAP-k2 cell line and assay. Cells weretreated with increasing concentrations of a p38 MAPK inhibitor (SB203580)in the presence of agonist (300-nM anisomycin). Comparable IC50 values,hill slopes, and signal windows were obtained on the two days. Each datapoint represents the percent maximal response measured from eightreplicate wells.

NuclearTraffickingAnalysisModule

QualitymetricsforHCS data

The Nuclear Trafficking Analysis Module provides amethod to quantitate the movement, in either direction,of the target molecules between the cytoplasm andthe nucleus. To do this, the quantitative image analysisroutine measures the fluorescence intensity of a targetmolecule in discrete nuclear and cytoplasmic regions,and then calculates the ratio of the sampled intensitiesas an index of translocation (Fig 8).

IN Cell Analyzer 3000 hardware and software combineto generate accurate, quantitative, robust, andreproducible assay results, which can be used to derivequality metrics that define how an assay is performingas it proceeds (Fig 9).

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Ordering information

Product Product code

IN Cell Analyzer 3000 instrument 25-8010-11(includes three analysis modules)

Object Intensity Analysis Module 63-0048-93

Nuclear Trafficking Analysis Module 63-0048-94

Plasma Membrane Trafficking Analysis Module 63-0048-95

Plasma Membrane Spot Analysis Module 63-0048-96

Granularity Analysis Module 63-0048-97

Cell Cycle Trafficking (Static) Analysis Module Available at end of 2003Morphology Analysis Module Available at end of 2003Granularity Update CypHer Analysis Module Available at end of 2003Apoptosis Analysis Module Available at end of 2003Neurite Extension Analysis Module Available at end of 2003

Table 2. Technical specifications.

Computer specifications 150 GB hard drive - Microsoft Windows 2000

Software control Graphical user interface for IN Cell Analyzer, Windows 2000 MFC application

Light sources Krypton laser: red (647 nm)(two lasers generate three Argon Laser: UV(364 nm) and green (488 nm)excitation laser-line wavelengths) Red, green, and blue LEDs (transmission mode)

Sample detection Selected wavelengths between 420 and 720 nm

High speed autofocus Dynamic, continuous infrared laser autofocus

Optics Automated optical pathway control with user controllable attenuation of excitation,band pass filters for each camera and scanning mode

Imaging Three high-speed, TE-cooled, 12-bit CCD cameras.Separate grayscale diagnostic camera

Field of view 0.75 × 0.75 mm, with 40× objective

Spatial resolution 1.2 µm

Scan time ≤ 2.5 min per 96-well microplate, at 2.4-µm resolution

Standard image size 1250 × 1250 pixels

Pixel size 0.6, 1.2, and 2.4 µm at high, medium, and low resolution respectively

Multiple field option Up to 30 individual fields can be imaged per well on a 96-well microplate, and up to9 fields can be imaged per well on a 384-well microplate

Dispenser 10 to 50 µl in 10-µl steps

Run capacity (unattended) 216 microplates, 96- or 384-well format

Sample chamber for Internal environment controlled at: 37 °C, ≥ 70% humidity, 5% CO2,live-cell kinetics (Ambient conditions are obtained when control is switched off)

Automation Compatible with externally scheduled automation packages

Plate format Designed to scan standard 96- or 384-well microplates

CypHer is a trademark of Amersham Biosciences Limited. Amersham and Amersham Biosciences are trademarks of Amersham plc.Windows is a trademark of Microsoft Corporation. © Amersham Biosciences UK Limited, 2003 - All rights reserved. All goods andservices are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group that suppliesthem. A copy of these terms and conditions is available on request. Amersham Biosciences UK Limited Amersham Place LittleChalfont Buckinghamshire England HP7 9NA. Amersham Biosciences AB SE-751 84 Uppsala Sweden. Amersham Biosciences Corp800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA. Amersham Biosciences Europe GmbH Munzinger Strasse 9 D-79111Freiburg Germany.

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