GE Healthcare

Cell integrity assaysHigh-content analysis of essential cell integrity andtoxicity parameters using the IN Cell Analysis System

In recent years the development of high-content analysishas allowed the development of a number of cellular assaysthat have the potential to provide information on potentialdrug toxicity earlier in the discovery process.

Cellular toxicity can occur through a diverse range ofmechanisms that disrupt cellular integrity. Membrane-soluble or pore-forming compounds may act directly on thecytoplasmic membrane and prevent the cell maintaininghomeostatic integrity, leading to necrosis. Other compoundsmay act indirectly to disrupt the cell’s biochemical,synthetic, or signaling integrity, leading to apoptosis. Furthercompounds may act directly or indirectly to damage thecell’s genetic integrity, resulting in inheritable mutation,disruption of proliferative integrity, or apoptosis.

Critical cell functions that can be analyzed using automatedand manual microscopes with GE Healthcare’s cellularreagent technologies and image analysis software in drug toxicity testing include:

• Membrane integrity

• Proliferative integrity

• Organelle integrity

• Nuclear integrity

• Genetic integrity

• Intracellular signaling integrity

Apoptotic cells show characteristic morphological andbiochemical features including nuclear and cytoplasmiccondensation, membrane blebbing, and membrane inversion.

In the early stages of apoptosis the anionic lipidphosphatidylserine (PS) translocates from the inner side ofthe plasma membrane to the outer layer. This inversion canbe imaged using fluorescently labeled annexin-V (a calcium-dependent phospholipid-binding protein) as a marker forearly apoptosis (Fig 2).

Analysis of cell morphology is a powerful and informativeadjunct to the use of fluorescent dyes for investigating toxicaction of candidate drugs. Analysis using cytoplasmic andnuclear shape descriptors (Fig 3A) allows rapid quantitationof cells exhibiting normal and aberrant morphology (Fig 3B)as a measure of drug effects on cellular integrity.

Cell toxicity and death caused by drugs can occur throughnecrosis or apoptosis. In some cases these events mayoccur sequentially or in parallel depending on the dose andduration of exposure of cells to a test compound. There areseveral morphological and biochemical differences betweennecrosis and apoptosis and these may be detected usinghigh-content analysis (HCA) markers (Table 1).

Necrosis typically occurs when cells are exposed to an injurythat damages the plasma membrane and prevents the cellfrom maintaining homeostasis. Necrosis can be readilydetected by imaging the uptake of cell-impermeablefluorescent dyes such as propidium iodide into cells withdamaged plasma membranes (Fig 1).

In contrast to passive necrosis, apoptosis is an active energyrequiring process that occurs under normal physiologicalconditions where cells are triggered to self-destruct.

Membrane integrity

Characteristics of necrosis Characteristics of apoptosis

Loss of plasma membrane integrity Membrane blebbing

Swelling of cytoplasm Shrinkage of cytoplasm and nucleus

Loss of homeostasis Alterations in membrane symmetry

Total cell lysis and dissolution of contents Cell fragmentation into smaller bodies

HCA markers for necrosis HCA markers for apoptosis

Propidium iodide uptake Annexin V binding

Increase in cell area Decrease in nuclear area

Decrease in cell number Chromatin condensation

Nuclear fragmentation and Increase in sub-nuclear objects

Table 1. Characteristics and HCA markers for necrosis and apoptosis.

Fig 1A. Intact (blue)and necrotic (red)cells identified bystaining withHoechst andpropidium iodiderespectively. Imageacquired on IN CellAnalyzer 1000.

Fig 1B. Induction of cellular necrosismeasured by uptakeof propidium iodidefollowing treatmentwith increasingconcentrations oftest compounds for 6 h.

Fig 3A. IN CellAnalyzer 1000cellular morphologyanalysis. Cellsshowing normal andapoptotic (arrow)morphology werecategorized byselecting individualcells withrepresentativemorphology (inset).

Fig 3B. Automatedclassification of cellmorphology. Fourautomaticallyselected parameters(selected features,top) were sufficientto separate the cellsin Figure 3A into twodistinct populations(scatterplot, bottom).The selectedapoptotic cell (arrow)is the same as thatidentified in Fig 3A.

Fig 2A. U2OS cellstreated with 20-μMstaurosporine for 24 hthen stained with 10-μM Hoechst, 10-μMpropidium iodide, and500-ng/ml FITC-labeled annexin V.Image acquired on INCell Analyzer 1000.Hoechst = blue,propidium iodide =red, and annexin V =green.

Fig 2B. Binding ofFITC-labeled annexinV to HeLa cellsincubated withincreasingconcentrations ofionomycin for 4 h.

Fig 4. HeLa cellsincubated (A) In theabsence of colchicine(B) In the presence ofcolchicine. Cells werefixed in ethanol,stained withpropidium iodide, andimaged on IN CellAnalyzer 1000 formeasurement of DNA content.

Proliferative integrity

The cell cycle is of key importance to many areas of drugdiscovery. This fundamental process provides on the onehand the opportunity to discover new targets for anticanceragents and improved chemotherapeutics, and on the otherhand requires the testing of drugs and targets in othertherapeutic areas for undesirable effects on the cell cycle.

Measurement of DNA content by flow cytometry of fixedcells stained with fluorescent dyes such as propidium iodideis a standard method of analyzing cell cycle distribution.Performing the same analysis using high-throughput imaging(Figs 4 and 5) provides a significant increase in throughputcoupled with the ability to multiplex cell cycle analysisdetermined by DNA content with other parameters.

Conventional immmunodetection procedures for detecting 5-bromo-2’-deoxyuridine (BrdU) incorporation into the DNA ofreplicating cells use acid or alkali denaturation to allowaccess of the anti-BrdU antibody. However, these methodscan significantly alter cell morphology and preclude the useof additional cellular probes.

To enable the use of BrdU assays for HCA, nucleasetreatment is applied during incubation with monoclonal anti-BrdU to allow antibody access without adversely affectingcell morphology or compromising the signal from multiplexedfluorescent probes.

Detection with a Cy™5-labeled second antibody allows BrdUincorporation to be multiplexed with GFP (Fig 6A) or analyzedwith other cellular markers.

For further in-depth analysis of the effects of compounds oncell cycle and proliferation, GE Healthcare has developed twodynamic cell cycle sensors based on EGFP fusion proteins(Fig 7). Coupled with automated image analysis modules,these G2/M and G1/S Cell Cycle Phase Markers (CCPMs) allowdetailed cell-by-cell analysis for effects of candidate drugs oncell cycle checkpoint progression, delay, and arrest. Imagingof CCPMs can be multiplexed with imaging of DNA contentand BrdU incorporation (Fig 6A) to yield a highly informativepicture of drug effects on the cell cycle (Fig 8).

Fig 5. DNA contenthistogram analysisof HeLa cells shownin Figures 4A and 4B.Cells incubated inthe presence ofcolchicine showsignificantaccumulation of cellsin G2 and M.

Fig 7. The G2/M CCPM (top) follows the expression and degradation ofcyclin B1 as a marker for cells transitioning from G2 to M. G2 cells(arrow) express the CCPM in the cytoplasm with the fusion proteinundergoing translocation to the nucleus in prophase and reachingmaximal intensity at mitosis. Destruction of the sensor post mitosis(under control of the cyclin B1 D-box) resets the sensor in G1 daughtercells (arrows) ready for a further cycle. The G1/S CCPM (bottom) followsthe subcellular location of DNA helicase B. Expression in M phase cellsis uniform (arrow) but segregates rapidly to nuclei in G1 daughter cells (arrows) with export to the cytoplasm as cells transition through S phase into G2 where the sensor is restricted to the cytoplasm.

Fig 8. Multiplexed analysis of DNA content, G1/S transition and BrdUincorporation. U2OS cells expressing the G1/S CCPM were incubated inthe presence and absence of a test compound and pulsed with BrdUfor 1 h prior to imaging on IN Cell Analyzer 1000. DNA content wasmeasured by staining with Hoechst nuclear dye and BrdUincorporation measured with the Cell Proliferation Fluorescence Assay.Each sphere represents data from a single cell, with CCPM datarepresented by the size of each sphere. In this assay treatment withcompound A induced an increase in DNA content from 2n/4n to 4n/8nwith associated mitotic by-pass resulting in a significant proportion of8n cells in G1 (large red spheres at 8nDNA).

Fig 6A. BrdUincorporationdetected with theCell ProliferationFluorescence Assayin G2/M Cell CyclePhase Markerexpressing U2OScells. Image acquiredon IN Cell Analyzer1000 (DNA, GFP,BrdU).

Fig 6B. BrdUincorporationmeasured in U2OScells in the presenceof increasingconcentrations ofmitomycin C.

Organelle integrity

Changes in the shape, distribution, or other characteristics ofsubcellular organelles can be an important indicator of toxicityin cellular assays. For example, swelling of mitochondriaaccompanies homeostatic disruption in the early stages of cellnecrosis, and leakage of proteins and other factors frommitochondria is an early indicator of apoptosis.

Use of the IN Cell Developer Toolbox (see page 15) allowspowerful procedures to be constructed for HCA usingfluorescent dye and protein organelle markers to detectchanges in fluorescence intensity, distribution, andmorphology accompanying toxicity (Fig 9).

Fig 9A. U2OS cells transiently expressing an Emerald-FPfusion protein targeted to mitochondria. Image acquired onIN Cell Analyzer 1000. Hoechst = blue, Emerald FP = green,and Mitotracker™ Red = red.

Fig 9B. Analysis of fusion protein expression and retentionin mitochondria using IN Cell Developer Toolbox.

Fig 10. Nuclear changes associated with drugtoxicity. Taxol-treated cells (bottom) showsignificant changes in nuclear morphologycompared with control cells (top) includingfragmentation (a) and swelling (b) as well as asignificant decrease in numbers ofnuclei/image. Images acquired on IN CellAnalyzer 1000.

Fig 11. Analysis of nuclear size andfragmentation in taxol-treated cells using IN CellAnalyzer Image Analysis Modules.

Nuclear integrity

Changes in the number, size, and shape of nuclei in HCAimages are a simple but powerful indicator of toxic effects incells exposed to test compounds. Decreases in nuclearnumber/image may indicate inhibitory effects on the cell cycleor may be due to loss of cells through lysis depending on theduration of exposure. Similarly, changes in nuclear size (Fig 10) may be indicative of cell cycle blockage in G2 (increasein nuclear size) or apoptotic cell death (decrease in nuclear sizewith chromatin condensation).

In the advanced stages of apoptosis many nuclei will showclear breakdown into two or more fragments (Fig 10). Theseparameters can readily be quantitated by HCA (Fig 11) using arange of IN Cell Analyzer Image Analysis Modules and can beapplied to any assay using a nuclear stain to gain valuableadditional information on compound toxicity.

Fig 12. Micronucleus formation during cell division.

Genetic integrity

Micronucleus induction is a key characteristic of genotoxiccompounds. Analysis of micronucleus formation is an importantcomponent of toxicology evaluation of new drug candidatesand other chemicals and materials, such as food dyes andcosmetics that are intended for human consumption or use, orwhich may be indirectly or accidentally consumed or ingested.

Micronuclei formation occurs during cell division of cellsexposed to genotoxic compounds either as a result of DNAstrand breakage (clastogenic compounds) or throughinterference with chromosome segregation (aneugeniccompounds) by interference with components of the cell’schromosome separation machinery, such as tubulin (Fig 12).

Manual scoring of micronucleus assays is time consuming andsubject to operator variance, bias, and error. Automatedanalysis of micronucleus assays allows significantly fasteranalysis and consistently objective scoring.

The IN Cell Analyzer Micronuclei Formation Analysis Moduleenables fast automated scoring of micronucleus assays. Thesoftware allows the user to set parameters to identify nuclei,segregate mono-nucleate and bi-nucleate cells (for cytokinesisblock protocols) based on nuclear DNA content and symmetry,and to define a search area around each nucleus to identifymicronuclei (Fig 13).

The software is compatible with either single-channel imaging(DNA staining only) or with two-channel imaging (DNA andcytoplasm staining). Additionally the software provides theoption to use a third imaging channel in combination with live-cell staining to detect and reject cells with damagedcytoplasmic membranes from assays where cytotoxicity is present.

In a typical cytokinetic block assay, exposure of cells toincreasing concentrations of compounds of knowngenotoxicity results in an increase in the percentage of bi-nucleate cells with micronuclei (Fig 14A). As cells are exposedto higher doses of compounds, cell cycle inhibition andcytotoxicity results in cell arrest prior to mitosis. This preventsmicronuclei formation, with a resulting drop in micronucleifrequency at higher compound doses (Fig 14B).

Fig 13. Identification of micronuclei using theMicronucleus Formation Analysis Module. (A) Hoechst stained nuclei (B) Segregation of bi-nucleate [B] and mono-nucleate cells [M], (C) Search boundaries used for detection ofmicronuclei (D) Micronuclei outlined in white.

Fig 14a. Micronucleus assay dose-responsecurves. CHO-K1 cells were exposed to increasingconcentrations of clastogens (Mitomycin C andBleomycin) and aneugens (Etoposide andDiethylstilbestrol) and micronuclei measured byautomated analysis.

Fig 14b. Micronuclei assay proliferationindices reporting the ratio of bi-nucleate tomono-nucleate CHO-K1 cells exposed toincreasing concentrations of clastogens andaneugens. Proliferation index measured byautomated analysis.

Intracellular signaling integrity

In addition to effects on the physical integrity of cells,candidate drugs may also interfere with essential cell signalingpathways. To allow evaluation of possible interactions with keyintracellular signaling pathways GE Healthcare has developedan extensive range of GFP translocation and nitroreductase(NTR) live-cell reporter gene assays packaged ready to use inadenoviral vectors. Ad-A-Gene Vectors are validated forfunction, provided in a convenient, ready to use format, andgive high-efficiency transduction in both established andprimary cell types (Fig 15).

Used alone or in combination with other cell integrity readoutsin HCA, Ad-A-Gene Vectors provide a powerful toolbox fordetailed investigation of toxic effects of candidate drugs oncellular integrity (Fig 16).

For further details of Ad-A-Gene Vectors and signalingpathway coverage, visit www.gehealthcare.com/ad-a-gene.

Fig 15. Cellular transduction with Ad-A-Gene Vectors.

Fig 16. Anisomycin-induced translocation ofGFP-MAPKAP-k2 fusion protein delivered toHeLa cells with Ad-A-Gene Vector.

IN Cell Investigator Software

The IN Cell Investigator software suite provides a comprehensivesolution to high-content image and data analysis by combiningthe latest versions of IN Cell Developer Toolbox and IN Cell AnalysisModules with Spotfire™ DecisionSite™ visualization software.

Investigator Analysis Modules are a range of preconfigured, fully validated, and quantitative image analysis routines thatgenerate statistically relevant data for over 50 applications. The modules are straightforward to use and deliver the mostrelevant measurements for the majority of assays including cellintegrity assays. Simply select the analysis required and startwork. You can either choose specific packages to suit yourunique requirements or combine multiple packages.

Investigator Developer Toolbox is designed for specialized high-content analysis applications where predeveloped imageanalysis is not suitable. The controlled and fully supportedenvironment helps biologists to build tailored, custom routinesenabling the user to rapidly analyze and interpret results ofcomplex and unique assays. A selection of advanced

segmentation, preprocessing, and post-processing tools providesfull control over the sequence of steps in analysis routines.

Together these image analysis options provide a wealth ofmultiparametric phenotypic data that provide deep insight intothe cellular integrity on many levels.

Spotfire DecisionSite is a powerful data analysis package thatenables rapid interactive visualization, filtering, and sorting ofhigh-content data. This allows the scientist to explore in-depthchanges to the cellular integrity in response to cellular stimuliand perturbation.

IN Cell TranslatorIN Cell Translator is an optional software tool to convert imagedata from other high-content imaging systems to the IN CellAnalyzer 1000 and 3000 format. This conversion allows theanalysis of images from other platforms with IN Cell Investigatorsoftware. Please contact us for a full list of compatible formats.

Fig 17. Spotfire DecisionSite 3D scatterplotof data from a siRNA screen. Data for cellnumber, nuclear area, and thenuclear/cytoplasmic distribution ratio ofthe G1S Cell Cycle Phase Marker EGFPfusion protein are shown as SD frommean for each siRNA knockdown. Datapoints are additionally coded fornuclear/cytoplasmic distribution by color,and for nuclear area by size.

Assays and reagentsProduct Pack size Code number

Ad-A-Gene VectorsEGFP-Glucocortocoid receptor 5 × 107 ifu GDS20008SMAD 9-EGFP 5 × 107 ifu GDS20004PLC-PH domain-EGFP 5 × 107 ifu GDS200022×FYVE domain-EGFP 5 × 107 ifu GDS20011STAT 3-EGFP 5 × 107 ifu GDS20009SMAD 2-EGFP 5 × 107 ifu GDS20003CRE-NTR 5 × 107 ifu GDS40001NFAT-RE NTR 5 × 107 ifu GDS40002Ubiquitin C-NTR 5 × 107 ifu GDS40003

This is a selection from a range of over 50 targets. Visit www.gehealthcare.com/ad-a-gene for the complete range of Ad-A-Gene Vectors.

Cell cycle productsG1S Cell Cycle Phase Marker Assay Screening* 25-9003-97G2M Cell Cycle Phase Marker Assay Screening* 25-8010-50Cell Proliferation Fluorescence Assay 500 wells 25-9001-89

* Research, technology evaluation, and non-profit assays are also available – please inquire.

CyDye™ labeled second antibodiesAnti-mouse IgG Cy2-Linked (from goat) 1 mg PA42002Anti-rabbit IgG Cy2-Linked (from goat) 1 mg PA42004Anti-mouse IgG Cy3-Linked (from goat) 1 mg PA43002Anti-rabbit IgG Cy3-Linked (from goat) 1 mg PA43004Anti-mouse IgG Cy5-Linked (from goat) 1 mg PA45002Anti-rabbit IgG Cy5-Linked (from goat) 1 mg PA45004

Image analysisProduct Code number

IN Cell Investigator Software, 1 license 28-4089-71IN Cell Investigator Software, 1 additional license 28-4089-75IN Cell Investigator Software, 5 concurrent licenses 28-4089-72IN Cell Translator Software 28-4047-40

Products for cellular integrity assays

GE Healthcare has developed a range of assays, reagents, and image analysis software that can be used to assess theeffects of candidate drugs on cellular integrity.

General Electric Company reserves the right, subject to any regulatory approval if required, to make changes inspecifications and features shown herein, or discontinue the product described at any time without notice orobligation. Contact your GE Representative for the most current information. © 2006 General Electric Company- All rights reserved. Redistribution is a trademark of BioImage A/S; CyDye and Cy are trademarks of GEHealthcare Companies Limited; Hoechst is a trademark of Hoechst AG. Mitotracker is a trademark of MolecularProbes. Spotfire and DecisionSite are trademarks of Spotfire Inc. Cyanine dyes are manufactured under licensefrom Carnegie Mellon University under patent number 5268486 and other patents pending. Use of productscontaining GFP is limited in accordance with the terms and conditions of sale. GFP Products are developed andsold under license from: BioImage A/S under patents US 6 172 188, EP 851874 and EP0986753 and otherpending and foreign patent applications. Invitrogen IP Holdings Inc (formerly Vertex Pharmaceuticals andAurora Biosciences Corporation) under US patents 5 625 048, 5 777 079, 5 804 387, 5 968 738, 5 994 077, 6 054321, 6 066 476, 6 077 707, 6 090 919, 6 124 128, 6 172 188, European patent 1104769 and Japanese patentJP3283523 and other pending and foreign patent applications. Columbia University under US patent Nos. 5 491084 and 6 146 826. University of Florida Research Foundation under patents US patents 5,968,750, 5,874,304,5,795,737, 6,020,192 and other pending and foreign patent applications; and Iowa Research Foundation. Rightsto use this product, as configured, are limited to internal use for screening, development and discovery oftherapeutic products; NOT FOR DIAGNOSTIC USE OR THERAPEUTIC USE IN HUMANS OR ANIMALS. No other rightsare conveyed. Ad-A-Gene Vectors are sold under license from: Advec Inc. under patent US 6 140 087, US 6 379943, US 6 756 226, US 6 855 534, and other pending and foreign patent applications. Transgene S.A is soldunder US 6 136 594 for internal research purposes only and not for any clinical, therapeutic, prophylactic,diagnostic or production use. The NTR Gene Reporter Assay is the subject of patent WO0186348 and otherpending and foreign patent applications, it is developed and sold under license from Cancer ResearchCampaign Technology limited and Proacta Therapeutics limited under patents US5633158, US5780585,US5977065, AU681337, AU725236 and other pending and foreign patent applications. NTR products are sold foruse with CytoCy5S in in vitro gene reporter assays only. Use in any in vivo application in humans or animals isstrictly prohibited. The G2M Cell Cycle Phase Marker Assay is the subject of patent applications AU 2002326036,CA 2461133, EP02760417.2, IL 160908, JP 2003-534582, and US 10/491762 in the name of AmershamBiosciences and Cancer Research Technology. The G1/S Cell Cycle Phase Marker Assay is the subject of patentapplications US 60/590814, US 60/645968 and 60/645915 in the name of Amersham Biosciences andVanderbilt University. The IN Cell Analyzer analysis modules are sold under license from Cellomics Inc. under USpatent No 6573039, 5989835, 6671624, 6416959, 6727071, 6716588, 6620591 6759206; Canadian patent No2328194, 2362117, 2,282,658; Australian patent No 730100 European patent 1155304 and other pending andforeign patent applications.

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