Celigo Demonstration Experiments - Nexcelom Bioscience · 2018-06-10 · Celigo Demonstration Experiment – NK cell-mediated cytotoxicity using calcein AM 9 1001321 Rev B Data Collection

CeligoDemonstrationExperiments

April2017 www.nexcelom.com/celigo

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Nexcelom's team of Field Applications Scientists, R&D Specialists and Product Managers are in frequent contact with researchers in the field, developing new experiments that can be performed on the Celigo image cytometer.

We have taken this information and generated three unique types of documents:

Celigo Assays: In these red-colored documents, we summarize in 1-2 pages a basic overview of how certain assays would be performed on the Celigo. We include any stains needed, the imaging channels used on the Celigo, and a truncated review of methods and results generated from the assay. These are quick snapshots, providing proof of concept of specific assays that have been performed successfully on the Celigo.

Celigo Demonstration Experiments: In these green-colored documents, we outline in detail specific experiments that have been conducted on the Celigo. In these demonstration experiments we highlight the plate set up, assay protocol, detailed results generated by the Celigo, as well as a conclusion and summary of the outcome of the experiment.

Celigo Application Protocols: In these blue-colored documents, we provide a step-by-step guide on how to prepare, perform and analyze specific assays using the Celigo image cytometer. These are designed to comprehensively walk users through every step of the experiment: from prep to data analysis.

Our team continues to create new Celigo Assays, Demonstration Experiments and Applications Protocols documents. For the most current materials, please visit our website or reach out to your local area manager or applications scientist. The chart on the next page depicts the Celigo Assays, Celigo Demo Experiments and Celigo Applications Protocols that currently exist for different applications and assays. If you are interested in viewing any of the Celigo Assays or Celigo Demo Experiment documents, they are individually readily available on www.nexcelom.com for download. Simply type the name of the assay into the search bar on the homepage. We have also created an eBook version of all our Celigo Assays and all our Celigo Application Protocols

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CeligoAssays

CeligoDemos

CeligoApplicationProtocols

Immuno-Oncology01_0001NKCellMediatedCytotoxicityw/CalceinAM X X X01_0002NKCellMediatedADCCw/CalceinAM X 01_0003NeutrophilMediatedADCCw/CalceinAM X 01_0004CDCw/CalceinAM X X01_0005ADCMediatedCytotoxicityw/BF X X01_0008CIKMediatedADCCw/CFSE&PI X 01_0009CARTTCellMediatedCytotoxicityw/CFSE&PI X 01_0010NK92CellMediatedADCCw/CalceinAM X X X01_0011PBMCMediatedCytotoxicityusingCalceinAM X X X FluorescentAssays02_0001CellCyclew/DAPI&BrdU X X02_0002Viabilityw/CalceinAM,Hoechest&PI X X02_0003EndpointApoptosisw/Caspase3/7&Hoechst X X X02_0004CellCyclew/EdU&DAPI X 02_0005EndpointViabilityw/DRAQ7&Hoechst X X X02_0006KineticViabilityw/DRAQ7 X X X02_0007HTViabilityusingAO/PI X X X02_0008HTViabilityusingAO/PI&BF X02_0009p53&phosphor-p53FLMarkerAnalysis X X02_0010EndpointViabilityw/PI&Hoechst X X X02_0011KineticViabilityw/PI X X X02_0012KineticApoptosisw/Caspase3/7 X X X02_0013CellCyclePIAnalysisusingA375cells X02_0014HPCproliferationmeasurementusingKi-67cellularmarker X X X02_0015Antibody-DependentReceptorInternalizationAssay X 02_0016Countandmeasurecellinfectivityofmicrocarriers X 02_0017AntibodyCellSurfaceBindingDetection X X02_0018GFPTransfectionEfficiencyMeasurement X 02_0019AntibodyDependentDrugUptakeAssay X 02_0020KineticpHrodoAntibodyInternalizationusingConfluence X X02_0021KineticAntibodyInternalizationusingpHrodoandBrightField X X02_0022EndpointAntibodyInternalizationusingpHrodoandHoechst X X 3DModels03_0001LabelFreeTumorSpheroidGrowthInhibition X X 03_00023DMulticellulartumorsphere(MCTS)invasionscreen X X X03_00033DMCTSSizeandMorphologyDeterminationAssay X03_00043DMCTSgrowthinhibitionscreening X X X03_00053DMCTSendpointapoptosisscreening X X X03_0006CountingofPatientDerivediDCOrganoids X 03_00073DMCTSEndpointViabilityScreeningAssay X X X03_00083DMCTSKineticPIAssay X X X Migration/Invasion04_0001TranswellMigrationw/DAPI X 04_0002ScratchWoundHealingAssayusingDirectCellCounting X X

5

04_0003ScratchWoundHealingAssayusingConfluence X X04_00042DCellMigrationAssaywithOrisPlatypusPlate X X X CellCounting05_0001Label-Freeadherentcellproliferationusingconfluence X X05_0002Label-Freesuspensioncellproliferationbydirectcellcounting X X BrightField CellLineDevelopment07_0001SingleCellDetectionofFACS-sortedcells X iPSCReprogramming

CeligoDemonstrationExperiment–NKcell-mediatedcytotoxicityusingcalceinAM 61001321 Rev B

AssayName:NKcell-mediatedcytotoxicityusingcalceinAMAssayID:Celigo_01_0001


Experiment:NKcell-mediatedcytotoxicityusingcalceinAM

Purpose MeasureNKcell-mediatedcytotoxicityusingcalceinAM-stainedK562andIMR32cells

CurrentMethod(s) CalceinReleaseAssay,FlowCytometryTargetCellType Target:K562,IMR32;Effector:NKcellsExperimentPlan ScanplateusingBrightFieldandGreenFluorescentchannelsHypothesis BymeasuringthechangesinthenumberofcalceinAM-positivecellsovertime,

the%cytotoxicitycanbecalculatedusingtime0andcontrolfornormalization

CeligoSetupPlateType Greiner65509096-wellblackwallclearbottomScanChannels BrightfieldandGreenResolution 1µm/pixelScanArea WholewellAnalysisMethod Target1+2ScanFrequency Hourly,upto4hoursScanTime ~6min


AssayProtocolandPlateSetup

Goal

MeasureNKcell-mediatedcytotoxicityusingcalceinAM-stainedK562andIMR32foradurationof4hours.

Protocol

Cellpreparation

1. K562andIMR32wereobtainedfromATCCandculturedinRPMI1640media2. DonorNKcellswereobtainedfrombuffycoatofPBMCsandexpandedbyco-culturingwithirradiated

(100Gy)K562Clone9.mbIL21andsupplementedwith50IU/mlIL23. TheK562andIMR32Targetcellswerestainedwith10µMofcalceinAM(Nexcelom,Cat#CS1-0119)for

30minandthenwashed3timeswithRPMImedia4. Thecellswerethenseededintoa96-wellplateat10,000cells/well5. Next,theEffectorNKcellswereaddedfollowingtheE:Tratiosontheplatemapbelow

a. ThespontaneousreleasesampleswerestainedTargetcellswithoutEffectorcellsb. ThemaximumreleasesampleswerestainedTargetcellswithTritonX-100


DataCollection

1. AfteraddingtheTargetandEffectorcells,theplatewascentrifugedtosettlethecellstothebottom2. Immediatelyafter,theplatewasscannedinCeligousingTarget1(Green)+2(BF)fort=0h3. Repeatedthescanningfort=1,2,3,and4h

DataAnalysis

• TheimagesforeachtimepointwereanalyzedtocounttotalnumberofcalceinAM-positivecellsineachwell

• ThesegmentationparametersinAnalyzeweresetupusingthecalceinAMfluorescentimagesacquiredatthelasttimepoint(t=4h)

o TheparameterswereappliedtotheothertimepointsforcountingcalceinAM-positivecells• MadesureonlythebrightcalceinAM-positivecellswerecounted

o Theparametersweresetuptonotcountpiecesofbrightdebriso Theparametersweresetuptonotcountdimcells

DataCalculation

• ThecalceinAM-positiveTargetcellsco-culturedwithEffectorcellswerecountedandrecorded• ThecalceinAM-positiveTargetcellsonlywerecountedandrecorded

• %𝐶𝑦𝑡𝑜𝑡𝑜𝑥𝑖𝑐𝑖𝑡𝑦 = 1 − -./012345067389:;<9;=-./01234506738>?@9:?A

• The%cytotoxicitywascalculatedforeverywellandaveragedtogenerateE:Tratio-dependentresponseandtime-coursemonitoring

CeligoDemonstrationExperiment–NKcell-mediatedcytotoxicityusingcalceinAM101001321 Rev B

Results1.Celigo-capturedcalceinAMfluorescentimagesatdifferentE:TratiosforIMR32

• BelowareexamplecalceinAMimagesforIMR32atdifferentE:Tratios• ThenumberofcalceinAM-positivecellsincreasedastheE:Tratiosdecreased

• Thebargraphshowsthe%cytotoxicityatdifferentE:TratiosanddifferenttimepointsforIMR32

• The%cytotoxicityincreasedastheE:Tratiosincreased• Similarly,the%cytotoxicityincreasedasthetimeincreased

0%10%20%30%40%50%60%70%80%90%

100%

10:1 5:1 2.5:1 1.3:1 0.6:1 0.3:1

%Cytotoxicity

E:TRatio

%NKcell-mediatedcytotoxicityofIMR32cells

t=1

t=2

t=3

t=4


• Thelinegraphshowsthe%cytotoxicityatdifferentE:Tratiosinrespecttoincubationtime

• The%cytotoxicityincreasedasthetimeincreasedforeachE:Tratio• AthigherE:Tratios,the%cytotoxicityalreadyreached~90%att=1h

0%10%20%30%40%50%60%70%80%90%

100%

1 1.5 2 2.5 3 3.5 4

%Cytotoxicity

IncubationTime(Hour)

Time-monitoringofNKcell-mediatedcytotoxicityofIMR32

10:1

5:1

2.5:1

1.25:1

0.6:1

0.3:1


2.Celigo-capturedcalceinAMfluorescentimagesatdifferentE:TratiosforK562• BelowareexamplecalceinAMimagesforK562atdifferentE:Tratios• ThenumberofcalceinAM-positivecellsalsodecreasedastheE:Tratiosincreased

• Thebargraphshowsthe%cytotoxicityatdifferentE:TratiosanddifferenttimepointsforK562

• The%cytotoxicityincreasedastheE:Tratiosincreased• Similarly,the%cytotoxicityincreasedasthetimeincreased

0%10%20%30%40%50%60%70%80%90%

100%

10:1 5:1 2.5:1 1.3:1 0.6:1 0.3:1

%Cytotoxicity

E:TRatio

%NKcell-mediatedcytotoxicityofK562

t=1

t=2

t=3

t=4


• Thelinegraphshowsthe%cytotoxicityatdifferentE:Tratiosinrespecttoincubationtime

• The%cytotoxicityincreasedasthetimeincreasedforeachE:Tratio• AthigherE:Tratios,the%cytotoxicityalreadyreached~90%att=1h

Conclusion• Time-coursetrackingofNKcell-mediatedcytotoxicitycaneliminatetheneedofmaximumrelease

control(TritonX-100)usedinreleaseassays,aswellasnormalizingtonon-uniformseedingatt=0h• TheCeligowasabletocountbothsuspensioin(K562)andadherent(IMR32)livecells• Thenumberofcellsusedissignificantlylessthanthecellsneededforreleaseassaysandflowcytometry

assays,whichcansavetime,moneyandpreciousprimaryimmunecellso Flowcytometryassaysandreleaseassaysusuallyrequireaseedingdensityof100,000Target

cells,whichtranslateto1millionEffectorcellsatE:Tratioof10:1o Celigorequireslessthan10,000Targetcells,whichtranslatetolessthan100,000Effectorcells

atE:Tratioof10:1• ThedecreaseinnumberoflivecalceinAM-positiveTargetcellsinthefluorescentimageswas

successfullymeasuredtoshowtheeffectofNKcell-mediatedcytotoxicity

0%10%20%30%40%50%60%70%80%90%

100%

1 1.5 2 2.5 3 3.5 4

%Cytotoxicity

IncubationTime(Hour)

Time-monitoringofNKcell-mediatedcytotoxicityofK562

10:1

5:1

2.5:1

1.3:1

0.6:1

0.3:1

CeligoDemonstrationExperiment–NKcell-mediatedADCCusingcalceinAM141001324 Rev B

AssayName:NKcell-mediatedADCCusingcalceinAMAssayID:Celigo_01_0010


Experiment:NKcell-mediatedADCCusingcalceinAM

Purpose Measureantibody-dependentNK92cell-mediatedcytotoxicityusingcalceinAM-stainedMDA-MB-231

CurrentMethod(s) LDHReleaseAssayTargetCellType Target:MDA-MB-231;Effector:NK92cellsExperimentPlan ScanplateusingBrightfieldandGreenFluorescentchannelHypothesis BymeasuringthechangesinthenumberofcalceinAM-positivecellsovertime,


CeligoSetupPlateType Greiner65509096-wellblackwallclearbottomScanChannels BrightfieldandGreenResolution 1µm/pixelScanArea WholewellAnalysisMethod Target1+2ScanFrequency Every2hours,upto6hoursScantime ~4min



Goal

MeasureADCCofNK92cell-mediatedcytotoxicityusingcalceinAM-stainedMDA-MB-231for6hours

Protocol

Cellpreparation

6. MDA-MB-231wereobtainedfromATCCandculturedinRPMI1640media7. NK92-CD16cellswerepurchasedandexpandedinRPMI1640media8. MDA-MB-231cellswerestainedwith10µMofcalceinAM(Nexcelom,Cat#CS1-0119)for30minand

thenwashed3timeswithRPMImedia9. Thecellswerethenseededintothe96-wellplateat10,000cells/well10. Next,NK92cellswereaddedat5:1E:Tratio11. Finally,antibodieswereaddedat0,1,10,100pg/ml;1,10,100ng/ml;1µg/ml;andcontrolantibody

a. ThespontaneousreleasesampleswerestainedTargetcellswithoutEffectorcells


DataCollection

1. Afteraddingthecellsandantibodies,theplatewascentrifugedtosettlethecellstothebottom2. Immediatelyafter,theplatewasscannedinCeligousingTarget1(Green)+2(BF)fort=0h3. Repeatedthescanningfort=2,4,and6h

DataAnalysis


• Usedthelasttimepointasabaselineforsettingupthegatingparameters• MadesureonlythebrightcalceinAMcellswerecounted

o Thepiecesofbrightdebrisweregatedouto Thedimcellsweregatedout

DataCalculation

4. ThecalceinAM-positiveTargetcellsco-culturedwithandwithoutEffectorcellswerecountedandrecorded

5. ThecalceinAM-positiveTargetcellswithandwithoutantibodieswerecountedandrecorded6. ThecalceinAM-positiveTargetcellsonlywerecountedandrecorded7. Normalizedtot=0

a. %𝐶𝑦𝑡𝑜𝑡𝑜𝑥𝑖𝑐𝑖𝑡𝑦 𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑𝑡𝑜𝑡𝑖𝑚𝑒 = 1 − -./012345067389JK-./012345067389JL

8. Normalizedtospontaneousreleasea. %𝐶𝑦𝑡𝑜𝑡𝑜𝑥𝑖𝑐𝑖𝑡𝑦 = %𝑐𝑦𝑡𝑜𝑡𝑜𝑥𝑖𝑐𝑖𝑡𝑦 𝑠𝑎𝑚𝑝𝑙𝑒 − %𝑐𝑦𝑡𝑜𝑡𝑜𝑥𝑖𝑐𝑖𝑡𝑦(𝑠𝑝𝑜𝑛𝑡)

9. The%cytotoxicitywascalculatedforeverywellandaveragedtogeneratedoseresponseandtimecoursemonitoring


Results1.Celigo-capturedcalceinAMfluorescentimagesatdifferenttimepoints

• Belowisanexampleofplateviewintheresultssectionshowingcellscountedontheplate

• BelowareexamplecalceinAMimagesforMDA-MB-231atdifferenttimepoints• ThenumberofcalceinAM-positivecellsdecreasedastimeincreased• ThepurpleoutlinesindicatethecountedcalceinAM-positivecells


• Dose-dependentcytotoxicityresultsatdifferenttimepoints

-2 0 2 4-1 0

0

1 0

2 0

3 0

4 0

5 0

6 0

lo g [A n tib o d y ] v s % L y s is

l o g [ A b ] , n g /m l

Ly

sis

(%

)

2h

4h

6 h

• Theresultsshowedincreasing%cytotoxicityastheantibodyconcentrationincreasedforeachtimepoint

2.Immunecomplexformation• BelowareexamplecalceinAMandbrightfieldoverlayimages,whichshowedtheformationofimmune

complexclustersatdifferenttimes

2h4h6h

%Cytotoxicity


• BelowareexamplecalceinAMandbrightfieldoverlayimagesat6hour,whichshowedtheformationofimmunecomplexclustersatdifferentantibodydosages

Conclusion• TheCeligowasabletomeasureADCCforNK92andMDA-MB-231bycountingthenumberofcalcein

AM-positivecellsovertime• ThenumberofcalceinAM-positivecellsdecreasedasantibodydosageandtimeincreased• Inaddition,theabilitytoviewbrightfieldimagesallowedobservationofformationofimmune

complexesindicatingcytotoxicity

CeligoDemonstrationExperiment–PBMC-mediatedcytotoxicityusingcalceinAM211001327 Rev B

AssayName:PBMC-mediatedcytotoxicyusingcalceinAMAssayID:Celigo_01_0011


Experiment:PBMC-mediatedcytotoxicityusingcalceinAM

Purpose MeasurePBMC-mediatedcytotoxicitybycountingtotalliveK562cellsovertimeinthepresenceandabsenceofIL2

CurrentMethod(s) ChromiumReleaseAssay,FlowCytometryTargetCellType Target:K562;Effector:PBMCsExperimentPlan ScanplateusingBrightfieldandGreenFluorescentchannelHypothesis BymeasuringthechangesinthenumberofcalceinAM-positivecellsovertime,


CeligoSetupPlateType Greiner65509096-wellblackwallclearbottomScanChannels BrightfieldandGreenResolution 1µm/pixelScanArea WholewellAnalysisMethod Target1+2ScanFrequency Hourly,upto4hoursScanTime ~7min



Goal

MeasurePBMC-mediatedcytotoxicityusingcalceinAM-stainedK562Targetcellsforadurationof4hours.

Protocol

Cellpreparation

12. K562cellswereobtainedfromATCCandculturedinRPMI1640media13. TwodonorswereobtainedfrombuffycoatusingFicollprocess14. TheK562Targetcellswerestainedwith5µMofcalceinAM(Nexcelom,Cat#CS1-0119)for30minand

thenwashed3timeswithRPMImedia15. Thecellwerethenseededintoa96-wellplateat10,000cells/well16. Next,thePBMCswereadded,followingtheE:Tratiosontheplatemapbelow17. Finally,IL2wasaddedtohalfofthewellstoactivateNKcells

a. ThespontaneousreleasesampleswerestainedTargetcellswithoutEffectorcellsb. ThemaximumreleasesampleswerestainedTargetcellswithTritonX-100


DataCollection

1. AfteraddingtheTargetandEffectorcellsandIL2,theplatewascentrifugedtosettlethecellstothebottom

2. Immediatelyafter,theplatewasscannedinCeligousingTarget1(Green)+2(BF)fort=0h3. Repeatedthescanningfort=1,2,3,and4h

DataAnalysis


• ThesegmentationparametersinAnalyzeweresetupusingthecalceinAMfluorescentimagesacquiredatthelasttimepoint(t=4h)

o TheparameterswereappliedtotheothertimepointsforcountingcalceinAM-positivecells• MadesureonlythebrightcalceinAM-positivecellswerecounted

o Theparametersweresetuptonotcountpiecesofbrightdebriso Theparametersweresetuptonotcountdimcells

DataCalculation

• ThecalceinAM-positiveTargetcellsco-culturedwithEffectorcellswerecountedandrecorded• ThecalceinAM-positiveTargetcellsonlywerecountedandrecorded• Normalizedtot=0

o %𝐶𝑦𝑡𝑜𝑡𝑜𝑥𝑖𝑐𝑖𝑡𝑦 𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑𝑡𝑜𝑡𝑖𝑚𝑒 = 1 − -./012345067389JK-./012345067389JL

• Normalizedtospontaneousreleaseo %𝐶𝑦𝑡𝑜𝑡𝑜𝑥𝑖𝑐𝑖𝑡𝑦 = %𝑐𝑦𝑡𝑜𝑡𝑜𝑥𝑖𝑐𝑖𝑡𝑦 𝑠𝑎𝑚𝑝𝑙𝑒 − %𝑐𝑦𝑡𝑜𝑡𝑜𝑥𝑖𝑐𝑖𝑡𝑦(𝑠𝑝𝑜𝑛𝑡)

• The%cytotoxicitywascalculatedforeverywellandaveragedtogenerateE:Tratio-dependentresponseandtime-coursemonitoring


Results1.Celigo-capturedcalceinAMfluorescentimagesatdifferentE:TratiosforK562

• BelowareexamplecalceinAMimagesforK562atdifferentE:Tratios• ThenumberofcalceinAM-positivecellsincreasedastheE:Tratiosdecreased

o SamplesinthepresenceofIL2showedhighercellkillingcomparedtosamplesinabsenceofIL2

• Thebargraphshowsthe%cytotoxicityatdifferentE:Tratiosfordifferentdonorsatt=4h

• The%cytotoxicityincreasedastheE:Tratiosincreased• SamplesinpresenceofIL2showedgreaterthandoublethecytotoxicityincomparisontosamplesin

absenceofIL2

-10%

0%

10%

20%

30%

40%

50%

60%

70%

80%

50 25 13 6 3 0

%Cytotoxicity

E:TRatios

E:TRatioResponseatT=4hour

Donor1- IL2

Donor2- IL2

Donor1+IL2

Donor2+IL2


2.Celigo-capturedcalceinAMfluorescentimagesatdifferenttimepointsforK562• BelowareexamplecalceinAMimagesforK562atdifferenttimepointsatE:Tratioof50:1• ThenumberofcalceinAM-positivecellsalsodecreasedasthetimeincreased

• Thelinegraphshowsthe%cytotoxicityinrespecttoincubationtime

• The%cytotoxicityincreasedasthetimeincreasedforeachdonoratE:Tratioof50:1• DonorPBMCswithactivationusingIL2showed%cytotoxicityashighas65%

-10%

0%

10%

20%

30%

40%

50%

60%

70%

80%

0 1 2 3 4 5

%Cytotoxicity

Time(hour)

Time-monitoringofPBMC-mediatedcytotoxicityofK562

Donor1- IL2

Donor1+IL2

Donor2- IL2

Donor2+IL2

TargetOnly


Conclusion• Time-coursetrackingofPBMC-mediatedcytotoxicitycaneliminatetheneedofmaximumrelease

control(TritonX-100)usedinreleaseassays,aswellasnormalizingtonon-uniformseedingatt=0h• Thenumberofcellsusedissignificantlylessthanthecellsneededforreleaseassaysandflowcytometry

assays,whichcansavetime,moneyandpreciousprimaryimmunecellso Flowcytometryassaysandreleaseassaysusuallyrequireaseedingdensityof100,000Target

cells,whichtranslateto1millionEffectorcellsatE:Tratioof10:1o Celigorequireslessthan10,000Targetcells,whichtranslatetolessthan100,000Effectorcells

atE:Tratioof10:1• ThedecreaseinnumberoflivecalceinAM-positiveTargetcellsinthefluorescentimageswas

successfullymeasuredtoshowtheeffectofPBMC-mediatedcytotoxicity

CeligoDemonstrationExperiment–EndpointapoptosisusingCaspase3/7withHoechst 281001318 Rev B

AssayName:EndpointapoptosisusingCaspase3/7withHoechstAssayID:Celigo_02_0003


Experiment:EndpointapoptosisassayusingCaspase3/7withHoechst

Purpose PerformapoptosisassayonMDA-MB-231andJurkatcellsCurrentMethod(s) FlowcytometryTargetCellType MDA-MB-231andJurkatcellsExperimentPlan ScanplateusingGreen,BrightfieldandBluechannelsHypothesis BymeasuringthenumberofCaspase3/7positivecells,wecandeterimethe

percentapoptoticcellsinthepopulation

CeligoSetupPlateType Greiner65509096-wellblackwallclearbottomScanChannels Green,BrightfieldandBlueResolution 1µm/pixelScanArea WholewellAnalysisMethod Target1+2+MaskScanFrequency EndPointScanTime ~15minutes

AssayProtocolandPlateSetupGoal:DetectandquantifyapoptoticcellsusingCaspase3/7andHoechststaininginadherentMDA-MB-231andsuspensionJurkatcelllines

Protocol

• SeedMDA-MB-231at10,000cells/wellandJurkatcellsat20,000cells/wellandallowtoincubateovernight• AddStaurosporineat3µMfinalconcentrationperwellandallowtoincubatefor4-6hours• Afterincubationiscompleted,prepareinPBSa2XconcentrationofCaspase3/7andHoechst

o Nexcelom,Cat#CSK-V0003-1• Remove100µLofmediafromallplatewells.• Add100µLof2XconcentrationofCaspase3/7andHoechstandincubatefor30minsat37°C• ImagetheplateusingtheCeligoimagecytometer

Platesetup

Seedingnumberofcells/well Drugtreatmentandcontrolwells

1 2 3 4 5 6 7 8 9 10 11 12AB 10000 10000 10000 10000 20000 20000 20000 20000C 10000 10000 10000 10000 20000 20000 20000 20000D 10000 10000 10000 10000 20000 20000 20000 20000E 10000 10000 10000 10000 20000 20000 20000 20000F 10000 10000 10000 10000 20000 20000 20000 20000G 10000 10000 10000 10000 20000 20000 20000 20000H

JurkatMDA-MB-231 StaurosporinDrugTreatment:1 2 3 4 5 6 7 8 9 10 11 12

AB 3µM 3µM 3µM 3µM 3µM 3µM 3µM 3µMC 3µM 3µM 3µM 3µM 3µM 3µM 3µM 3µMD 3µM 3µM 3µM 3µM 3µM 3µM 3µM 3µME Control Control Control Control Control Control Control ControlF Control Control Control Control Control Control Control ControlG Control Control Control Control Control Control Control ControlH


ResultsDrug-treatedMDA-MB-231andJurkatcellsshowedanincreaseinCaspase3/7positivecells

• TotalnumberofnucleatedcellswasdeterminedbycounterstainingthecellswithHoechst• Totalnumberofapoptoticcellswasdeterminedbycountingthenucleatedcellsstainedwithgreen

Caspase3/7reagent• Determinedthepercentofapoptotic-positivecells

Plate-LevelDataViewallowsaquickobservationofthetotalnumberofcellsandgreenCaspase3/7positivecells,aswellaspercentCaspase3/7positivecells.CurrentlydisplayingpercentCaspase3/7.

Whole-wellviewallowshighresolutionobservationofimagesandatzoomedlevels.

Whole-WellViewCaspase3/7cells

Zoomed-InViewCaspase3/7cells

MDA-MB-231Adherent JurkatSuspension

Control

3µM

Staurospo

rine


GatePlotsforCaspase3/7PositveCells:ApoptosisCaspase3/7gatinganalysisofaMDA-MB-231andJurkatcells

CaspasePos

CaspaseNeg

Control

CaspasePos

CaspaseNeg3µM

Staurospo

rine

CaspasePos

CaspaseNeg

Control

CaspasePos

CaspaseNeg

3µM

Staurospo

rine

MDA-MB-231Adherent:

JurkatSuspension:


Graphs

• Cellcountsforadherent,MDA-MBA-231andsuspension,Jurkatcellsplotted• UsingHoechstcellcountsastotal,percentCaspase3/7positivecellsareareplottedonbargraphs

Conclusion• TheCeligosuccessfullyperformedCaspase3/7apoptosisassayusingMDA-MB-231andJurkatcell

lines• Acquisitionofhighresolutionbrightfield,Caspase3/7,andHoechstfluorescentimagesofanentire

96wellplatetook~15minutes• PerforminganendpointapoptosisassayusingCaspase3/7withHoechstallowsfortheenumeration

ofthetotalnumberofnucleatedcellsandtotalnumberofCaspase3/7positivecells,aswellastodeterminethepercentofapoptosis.

CeligoDemonstrationExperiment–EndpointviabilityusingDRAQ7andHoechst 331001360 Rev B

AssayName:EndpointviabilityusingDRAQ7andHoechstAssayID:Celigo_02_0005


Experiment:EndpointviabilityusingDRAQ7andHoechst

Purpose PerformendpointviabilityassayonMDA-MB-231andK562cellstreatedwithBenzethoniumfor24,48and72hours

CurrentMethod(s) CellTiterGlo,FlowCytometryTargetCellType MDA-MB-231adherentandK562suspensioncellsExperimentPlan ScanplateusingFarRed,BrightfieldandBluechannelsHypothesis DrugtreatmentwillincreasethepercentageofDRAQ7-positivecells

CeligoSetupPlateType Greinercat#781091384-wellblackwallclearbottomScanChannels FarRed,BrightfieldandBlueResolution 1µm/pixelScanArea WholewellAnalysisMethod Target1+2+MaskScanFrequency Daily,for3daysScanDuration ~15minutes

AssayProtocolandPlateSetupGoal:DetectandquantifythedeadcellsusingDRAQ7andHoechsttotalstaininadherentMDA-MB-231andsuspensionK562celllines

Protocol

• SeededMDA-MB-231andallowedtoincubateovernight.SuspensionK562cellswereplatedonthefirstdayoftheexperiment

• PreparedandseriallydilutedthedrugBenzethoniumtogenerateadoseresponse• Preparedthecontrolwithwaterinmedia• Addeddrugdoseresponseandcontroltothewellsaccordingtotheplatemap• Incubatedtheplatefor24,48and72hours• PreparedadyemixsolutionofDRAQ7andHoechstinPBS• Addeddyemixtothedrugtreatedwellsandincubatedtheplate• ImagedtheplateusingCeligoimagecytometer


PlatemapsforBenzethonium(µM)drugtreatmentandHoechsttimepointstaining:

Results

Drug-treatedMDA-MB-231andK562cellsshowedanincreaseinDRAQ7-positivecells• DRAQ7-positivecells(deadcells)weredeterminedattheendpointof24,48and72hoursbyDRAQ7and

Hoechstasatotalnucleatedstain

GatePlotsforDRAQ7PositveCells:ExampleofgatingsettingsforHoechstandDRAQ7-stainedMDA-MB-231adherentcellline,withbluegraphicoverlayoutliningallobjectsandredgraphicoverlayoutliningDRAQ7cells.Similarsetupwasfollowedtoworkwithsuspensioncells.

NegativeControl

DrugTreatmentofBenzethonium13 14 15 16 17 18 19 20 21 22 23 24

ABCDEFGHIJKLMNOP

Control

6.7

5.2

4.0

3.1

2.4

25.0

19.2

14.8

11.4

8.8

Control

25.0

19.2

14.8

11.4

8.8

6.7

5.2

4.0

3.1

2.4

Control

6.7

5.2

4.0

3.1

2.4

25.0

19.2

14.8

11.4

8.8

HoechstStainEndpoint:ABCDEFGHIJKLMNOP

Hoechstaddedat24hrtimepoint



PositiveControl

DRAQ7(+)


CeligoproducedthefollowingresultsforMDA-MB-231andK562:percentdeadcellsafter24hoursofBenzethoniumdrugtreatment

Graphing1. GraphsweregeneratedusingGraphPadPrismforthedoseresponseofBenzethoniumafter24,48

and72hoursoftreatment.Inthisexperiment,theaverageof4datapointswereplotted.2. Theresultsareshownbelow:

• IC50valueswerecalculatedusingGraphPadPrism

Conclusion• DrugtreatedMDA-MB-231andK562celllinesweresuccessfullyimagedandanalyzedonCeligo• EndpointviabilityassayusingDRAQ7andHoechstallowedfortheenumerationoftotalcountsand

percentagesofDRAQ7-positivecells• AcquisitionofhighresolutionDRAQ7andbrightfieldimagesof384-wellplatetookabout15

minutes

JurkatSuspension:

MDA-MB-231Adherent K562Suspension

CeligoDemonstrationExperiment–KineticViabilityusingDRAQ7 371001361 Rev B

AssayName:KineticviabilityusingDRAQ7AssayID:Celigo_02_0006


Experiment:KineticviabilityusingDRAQ7

Purpose PerformkineticviabilityassayonMDA-MB-231andK562cellsCurrentMethod(s) CellTiterGlo,FlowCytometryTargetCellType MDA-MB-231adherentandK562suspensioncellsExperimentPlan ScanplateusingFarRedandBrightfieldchannelsHypothesis DrugtreatmentwillincreasethepercentageofDRAQ7-positivecellsovertime

CeligoSetupPlateType Greinercat#781091384-wellblackwallclearbottomScanChannels FarRed,BrightfieldResolution 1µm/pixelScanArea WholewellAnalysisMethod Target1+2ScanFrequency Daily,for3daysScanDuration ~15minutes

AssayProtocolandPlateSetupGoal:DetectandquantifydeadcellsusingDRAQ7staininadherentMDA-MB-231andsuspensionK562celllines

Protocol

• SeededMDA-MB-231at2,000cells/wellandallowedtoincubateovernight• SuspensioncellswereplatedthedayofexperimentwithworkingsolutionofDRAQ7at3,000cells/well• PreparedthedrugBenzethoniumandseriallydilutedtogeneratedoseresponse• Preparedcontrolwithwaterinmedia• Removedthemediafromthewellsofadherentcells• Addeddrugdoseresponseandcontroltobothadherentandsuspensioncells• AddedDRAQ7toadherentcellsonly• Incubatedtheplatefor24,48and72hourswithdruganddye• ImagedtheplateusingtheCeligoimagecytometer


PlatemapforBenzethonium(µM)drugtreatmentandDRAQ7timepointstaining

ResultsDrug-treatedMDA-MB-231andK562cellsshowedanincreaseinDRAQ7-positivecells

• DRAQ7-positivecellsweredeterminedbystainingthecellsfor24,48and72hours

ImagesandfluorescentobjectidentificationlookedasshownbelowforDRAQ7-stainedcells“GraphicOverlay”segmentation

BF+FarRedImage FarRedImage

K562

Suspe

nsion

FarRedGraphicOverlay BFImage

MDA

-MB-23

1Ad

herent


ResultsfortheMDA-MB-31andK562countsofdeadcellsafter24hoursofBenzethoniumdrugtreatment

Graphs

3. GraphsweregeneratedusingGraphPadPrismforthedoseresponseofBenzethoniumafter24,48and72hourstreatment.Inthisexperiment,theaverageof4datapointswereplotted.

4. IC50valueswerecalculatedusingGraphPadPrism§ CelldeathincreaseovertimewithBenzethonium(14.5µM)versusthecontrol

Conclusion• DrugtreatedMDA-MB-231andK562celllinesweresuccessfullyimagedandanalyzedonCeligo• KineticviabilityassayusingDRAQ7allowsfortheenumerationoftotalcountsofDRAQ7-positive

cellsoverthetime• AcquisitionofhighresolutionDRAQ7andbrightfieldimagesof384-wellplatetookabout15

minutes


CeligoDemonstrationExperiment–HTsuspensioncellcountandviabilityusingAO/PI 411001369 Rev B

AssayName:HTsuspensioncellcountandviabilityusingAO/PIAssayID:Celigo_02_0007


Experiment:High-throughputsuspensioncellcountandviabilityusingAO/PI

Purpose MeasureJurkatcellconcentrationandviabilityattitrationsofconcentrationsanddifferentmixtureofcellviability,whichcandemonstratethehigh-throughputcapabilityofCeligoimagecytometer

CurrentMethod(s) Manualcounting;singlesampleautomatedcellcountingTargetCellType JurkatcellsExperimentPlan ScanplateusingtheGreenandRedFluorescentchannelsHypothesis BymeasuringthetitrationofcellconcentrationsusingAO/PIaswellascell

viability,thelinearityofthemethodcanbedetermined

CeligoSetupPlateType Greiner67509096-wellblackwallclearbottomhalfareaplateScanChannels Green,RedResolution 1µm/pixelScanArea WholewellAnalysisMethod Target1+2ScanFrequency EndpointScanTime ~7min



Goal

MeasureJurkatcellconcentrationandviabilityatatitrationofcellconcentrationandviabilitytodeterminelinearityofthemethod.Themethodcanimprovethespeedandefficientofcellcounting,wherenumeroussamplesarerequiredtobecountedonadailybasisforbioprocessing,PBMCssamples,orsimplycellculture.

Protocol

Stainpreparation

1. DilutedAO/PIstain(Nexcelom,Cat#CS2-0106)by10XinPBStotheworkingconcentration2. Pipetted25µLoftheAO/PIstainintoeachwell

Cellpreparation

18. JurkatcellswereobtainedfromATCCandculturedinRPMI1640mediawithFBS19. Jurkatcellswerecollected,dilutedinPhosphateBufferedSaline(PBS),countedandbroughttoastarting

concentrationof~1x107cells/ml

Plate1Preparation:Serialdilutionforlinearity

1. TheJurkatcellsamplewasserialdiluted2Xto2,000Xwithastartingconcentrationof~1x107cells/ml2. EachJurkatcellconcentration(5µL)wasthenpipettedintotheplateaccordingtotheplatemapshown

below

Dilution 1 2 3 4 5 6 7 8 9 10 11 12A Neat 0.5 0.25 0.125 0.0625 0.0313 0.0156 0.0078 0.0039 0.0020 0.0010 0.0005B Neat 0.5 0.25 0.125 0.0625 0.0313 0.0156 0.0078 0.0039 0.0020 0.0010 0.0005C Neat 0.5 0.25 0.125 0.0625 0.0313 0.0156 0.0078 0.0039 0.0020 0.0010 0.0005D Neat 0.5 0.25 0.125 0.0625 0.0313 0.0156 0.0078 0.0039 0.0020 0.0010 0.0005E Neat 0.5 0.25 0.125 0.0625 0.0313 0.0156 0.0078 0.0039 0.0020 0.0010 0.0005F Neat 0.5 0.25 0.125 0.0625 0.0313 0.0156 0.0078 0.0039 0.0020 0.0010 0.0005G Neat 0.5 0.25 0.125 0.0625 0.0313 0.0156 0.0078 0.0039 0.0020 0.0010 0.0005H Neat 0.5 0.25 0.125 0.0625 0.0313 0.0156 0.0078 0.0039 0.0020 0.0010 0.0005


Plate2Preparation:ViabilityassessmentwithAO/PI

1. HalfoftheJurkatcellswereheat-killedinboilingwaterfor15minutes2. Afterheat-killing,theJurkatcellsweremixedwithfreshJurkatcellstoproduce5differentviability

samplesof0,25,50,75,and100%3. Jurkatcellsateachviabilitypercentagewerepipettedintotheplateaccordingtotheplatemapshown

below

Viability 1 2 3 4 5A 100% 75% 50% 25% 0%B 100% 75% 50% 25% 0%C 100% 75% 50% 25% 0%D 100% 75% 50% 25% 0%E 100% 75% 50% 25% 0%F 100% 75% 50% 25% 0%G 100% 75% 50% 25% 0%H 100% 75% 50% 25% 0%

DataCollection

4. AfteraddingtheJurkatcells,theplatewascentrifugedtosettlethecellstothebottom5. Immediatelyafter,theplatewasscannedinCeligousingTarget1(Green)+2(Red)foranendpointscan

a. Thismethodisusedinsteadofthe“CellViability”applicationbecausetheresultsareexporteddirectlyintoExceltocalculatetheconcentrationandviability

DataAnalysis

• TheimagesforeachJurkatcellconcentrationandviabilitywereanalyzedtocounttotalnumberofAOandPIpositivecellsinthewells

• ThesegmentationparametersintheAnalyzetabweresetupusingAOandPIfluorescentimages,whichwereusedtoanalyzetheentireJurkatcelltitrationandviabilitymicroplate

• MadesureonlythebrightAOandPIpositivecellswerecountedo Theparametersweresetuptonotcountpiecesofbrightdebris

DataCalculation

• TheAOandPIpositivecellswerecountedandtheresultswereexportedtoExcelforeachplate• Usedthefollowingtwoequationstocalculateconcentrationandviabiltiy

a. 𝐶𝑒𝑙𝑙𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 = -1//Q067381RS.SSU

𝑐𝑒𝑙𝑙𝑠/𝑚𝐿,where0.005mLwasthevolumeofcellspipettedintothewell

b. 𝑉𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦% = 4Z067381R4Z[\]067381R

𝑥100• Theconcentrationandviabilitywerecalculatedforeachwell


Results1.Celigo-capturedJurkatcellconcentrationseriesAOfluorescentimages

• BelowareexampleAOfluorescentimagesatdifferentconcentrations


• The Jurkat cell concentrations measured using the Celigo were graphed in a correlation plot to thetheoreticalconcentrationscalculatedfromthedilutionfactors

• Theresultsshowedhighlinearcorrelation(R2value=0.9937)betweenthemeasuredcellconcentrationcomparedtothetheoreticalconcentrations

• TheCeligocanmeasurefrom1cell/welltoapproximately1x107cells/ml• Bypipettingsmallervolumeofcellsintoeachwell,themaximumconcentrationlimitcanincrease


2.Celigo-capturedJurkatcellviabilityseriesAO/PIfluorescentimages• BelowareexampleAO/PIfluorescentimagesatdifferentviabilities• ThenumberofAOpositivecellsdecreasedastheviabilitydecreased• ThenumberofPIpositivecellsincreasedastheviabilitydecreased


• TheJurkatcellviabilitiesmeasuredusingtheCeligowereplotted inrespecttothetheoreticalviabilityfromthedifferentfreshandheat-killedJurkatmixture

• The results showed high linear correlation (R2 = 0.9977) between themeasured cell viability and thetheoreticalviability

• TheCeligocanmeasurefrom0to~100%ofviability

Conclusion• High-throughputcellcountingandviabilitycanbeachievedusingtheCeligoimagecytometer• TheCeligowasabletomeasure2fluorescentchannelsinone96-wellplateinapproximately7minusing

AO/PI• TheCeligowasabletoshowhighlinearresultsformeasuringcellconcentrationsandviability

o Forconcentration,theCeligocancountfrom1celltoapproximately1x107cells/mlo Forviability,theCelgiocanmeasureviabilityfrom0to100%

• TheCeligoimagecytometryprovidesarapidandsimplemethodforhigh-throughputcellcountingandviabilityforsuspensionscells

CeligoDemonstrationExperiment–EndpointviabilityusingPIandHoechst 491001355 Rev B

AssayName:EndpointviabilityusingPIandHoechstAssayID:Celigo_02_0010


Experiment:EndPointViabilityAssayUsingPIandHoechst

Purpose PerformendpointviabilityassayonMDA-MB-231andK562cellstreatedwithBenzethoniumfor24,48and72hours

CurrentMethod(s) CellTiterGlo,FlowcytometryTargetCellType MDA-MB-231adherentandK562suspensioncellsExperimentPlan ScanplateusingRed,BrightfieldandBluechannelsHypothesis DrugtreatmentwillincreasethepercentageofPI-positivecellsovertime

CeligoSetupPlateType Greiner781091384-wellblackwallclearbottomScanChannels Red,BrightfieldandBlueResolution 1µm/pixelScanArea WholewellAnalysisMethod Target1+2+MaskScanFrequency Daily,upto3daysScanDuration ~15minutes


Goal:DetectandquantifydeadcellsusingPIandHoechststainsinadherentMDA-MB-231andsuspensionK562celllines

Protocol

• SeededMDA-MB-231andallowedtoincubateovernight.K562Suspensioncellswereplatedonthedayoftheexperiment

• PreparedandseriallydilutedthedrugBenzethoniumtogenerateadoseresponse• Preparedthecontrolwithwaterinmedia• Addeddrugdoseresponseandcontroltothewells• Incubatedtheplatefor24,48and72hours• PreparedadyemixsolutionofPIandHoechstinPBS• Addeddyemixtothedrug-treatedwellsandincubatedtheplate• ImagedtheplateusingCeligoimagecytometer


PlatemapsforBenzethonium(µM)drugtreatmentandHoechsttimepointstaining:

Results

Drug-treatedMDA-MB-231andK562cellsshowedanincreaseinPI-positivecells• PI-positivecellsweredeterminedbystainingthecellsattheendpointof24,48and72hourswithPI

andHoechststains

GatePlotsforPI-PositveCells:ExampleofgatingsettingsforHoechst+PIstainedMDA-MB-231adherentcellline,withBluegraphicoverlayoutliningallobjectsandRedgraphicoverlayoutliningPI-positivecells.Followasimilarsetupforworkwiththesuspensioncells.

DrugTreatmentofBenzethonium(µM)1 2 3 4 5 6 7 8 9 10 11 12 13

ABCDEFGHIJKLMNOP

25.0

19.2

14.8

11.4

8.8

Control

6.7

5.2

4.0

3.1

2.4

Control

25.0

19.2

14.8

11.4

8.8

6.7

5.2

4.0

3.1

2.4

Control

6.7

5.2

4.0

3.1

2.4

25.0

19.2

14.8

11.4

8.8

25µMBenzethonium

NegativeControl

DeadPI+

DeadPI+


CeligoproducedthefollowingresultsfortheMDA-MB-231andK562countsofpercentdeadcellsafter24hoursofBenzethoniumdrugtreatment

Graphing• GeneratedagraphusingMicrosoftExcelcomparing25µMBenzethoniumtothecontrolafter24,48

and72hourstreatment.Inthisexample,theaverageof4datapointswereplotted.

• IC50valueswerecalculatedusingGraphPadPrism.

Conclusion• TheCeligosuccessfullyperformedPIviabilityassayusingMDA-MB-231andK562celllines• PerforminganendpointviabilityassayusingPIandHoechstallowedforthecalculationof

percentagesfromtheenumerationofPI-positiveandtotalcellcounts• AcquisitionofhighresolutionPIandbrightfieldimagesofa384-wellplatetook~15minutes

JurkatSuspension:

ControlBenzethoniumDoseResponse

ControlBenzethoniumDoseResponse

K562Suspension MDA-MB-231Adherent

CeligoDemonstrationExperiment–KineticviabilityusingPI 531001358 Rev B

AssayName:KineticviabilityusingPIAssayID:Celigo_02_0011


Experiment:KineticviabilityusingPropidiumIodide

Purpose PerformkineticviabilityassayonMDA-MB-231andK562cellsCurrentMethod(s) CellTiterGlo,FlowCytometryTargetCellType MDA-MB-231adherentandK562suspensioncellsExperimentPlan ScanplateusingRedandBrightfieldchannelsHypothesis DeterminethecountsofPI-positivecellskineticallyat24,48and72hours

CeligoSetupPlateType Greiner781091384-wellblackwallclearbottomScanChannels Red,BrightfieldResolution 1µm/pixelScanArea WholewellAnalysisMethod Target1+2ScanFrequency Daily,for3daysScanDuration ~15minutes

AssayProtocolandPlateSetupGoal:DetectandquantifydeadcellsusingPIstaininadherentMDA-MB-231andsuspensionK562celllines

Protocol

• SeededMDA-MB-231at2,000cells/wellandandallowedtoincubateovernight• Suspensioncellswereplatedthedayofexperimentwithworkingsolutionof2XPIat3,000cells/well• PreparedBenzethoniumat25µMfinalconcentrationandseriallydilutedby1.3dilutionfactor• Removedmediaandaddeddrug,controlandPIstaintowells• Incubatedtheplatefor24,48and72hourswithdruganddye• ImagedtheplateusingtheCeligoimagecytometer

PlatemapforBenzethonium(µM)drugtreatmentandPIstaining


ResultsDrug-treatedMDA-MB-231andK562cellsshowedanincreaseinPIpositivecells

• PI-positivecellsweredeterminedbystainingthecellsfor24,48and72hours

TypicalimagesandfluorescentobjectidentificationlookedasshownbelowforPIstainedcells“GraphicOverlay”segmentation

ResultsfortheK562andMDA-MB-31countsofdeadcellsafter24hoursofBenzethoniumdrugtreatment


BF+RedImage RedImage BFImageImages+GraphicOverlay

K562

-Su

spen

sion

MDA

-MB-23

1-A

dheren

t


Graphs

5. GeneratedagraphusingMicrosoftExcelcomparing25µMBenzethoniumtothecontrolafter24,48and72hoursoftreatment.Inthisexample,theaverageof4datapointswereplotted

• IC50valueswerecalculatedwithGraphPadPrism

• CelldeathincreasedovertimewithBenzethonium(14.5µM)versusthecontrol

Conclusion• TheCeligosuccessfullyperformedPIviabilityassayusingMDA-MB-231andK562celllinesdrug

treatedwithBenzethonium• PerformedkineticviabilityassayusingPIallowedfortheenumerationoftotalnumberofPI-positive

cellsoveraperiodof24,48and72hours

CeligoDemonstrationExperiment–KineticApoptosisusingCaspase3/7 571001388 Rev A

AssayName:KineticapoptosisusingCaspase3/7AssayID:Celigo_02_0012


Experiment:KineticapoptosisassayusingCaspase3/7

Purpose PerformapoptosisassayonMDA-MB-231andJurkatcellsCurrentMethod(s) FlowcytometryTargetCellType MDA-MB-231andJurkatcellsExperimentPlan ScanplateusingGreenandBrightfieldchannelsHypothesis BymeasuringthenumberofCaspase3/7positivecells,wecandeterminethe

countsofapoptoticcellsinthepopulation

CeligoSetupPlateType 96-wellGreiner655090blackwallclearbottomScanChannels GreenandBrightfieldResolution 1µm/pixelScanArea WholewellAnalysisMethod Target1+2ScanFrequency 0h,2h,4h,6hand8hoursScanTime ~15minutes

AssayProtocolandPlateSetupGoal:DetectandquantifyapoptoticcellsusingCaspase3/7staininginadherentMDA-MB-231andsuspensionJurkatcelllines

Protocol

• SeededMDA-MB-231at10,000cells/wellandallowedtoincubateovernight• SeededJurkatcellsat20,000cells/wellonthedayofexperiment• AddedStaurosporineat3µMfinalconcentrationandCaspase3/7substrateat4µMfinalconcentrationper

wellandallowedtoincubatefor8hoursat37°C• ImagedtheplateeverytwohoursusingtheCeligoimagecytometerforatotalof8hours

Platesetup

Seedingnumberofcells/well Drugtreatmentandcontrolwells

1 2 3 4 5 6 7 8 9 10 11 12AB 10000 10000 10000 10000 20000 20000 20000 20000C 10000 10000 10000 10000 20000 20000 20000 20000D 10000 10000 10000 10000 20000 20000 20000 20000E 10000 10000 10000 10000 20000 20000 20000 20000F 10000 10000 10000 10000 20000 20000 20000 20000G 10000 10000 10000 10000 20000 20000 20000 20000H

JurkatMDA-MB-231 StaurosporineDrugTreatment:1 2 3 4 5 6 7 8 9 10 11 12

AB 3µM 3µM 3µM 3µM 3µM 3µM 3µM 3µMC 3µM 3µM 3µM 3µM 3µM 3µM 3µM 3µMD 3µM 3µM 3µM 3µM 3µM 3µM 3µM 3µME Control Control Control Control Control Control Control ControlF Control Control Control Control Control Control Control ControlG Control Control Control Control Control Control Control ControlH


ResultsDrug-treatedMDA-MB-231andJurkatcellsshowedanincreaseinCaspase3/7positivecells

• Brightfieldimageswerecapturedtomonitorcellhealthandmorphology• ThetotalnumberofapoptoticcellswasdeterminedbycountingthecellsstainedwithgreenCaspase

3/7reagent

Plate-LevelViewallowsforquickobservationsofthetotalnumberofgreenCaspase3/7positivecells.Shownbelowaretypicalresultsofapoptotic(Caspase3/7positive)cellsafter8hoursofdrugtreatment.

Whole-wellviewallowsforobservationofhighresolutionimages.

Whole-WellViewCaspase3/7cells

Zoomed-InViewCaspase3/7cells

MDA-MB-231Adherent JurkatSuspension

Control

3µM

Stau

rosporine


Graphs

6. InMicrosoftExcel,createaveragesandstandarddeviationsofthecontrolanddrug-treatedwells.

7. Generatea“Bargraph”comparing3µMStaurosporinetothecontrolover8hourtimecourse.Inthisexample,theaverageof12datapointswereplotted.


Conclusion• TheCeligosuccessfullyperformedCaspase3/7apoptosisassayusingMDA-MB-231andJurkatcell

lines• AcquisitionofhighresolutionbrightfieldandgreenCaspase3/7fluorescentimagesofanentire96

wellplatetook~15minutes• PerformingkineticapoptosisassayusingCaspase3/7allowsfortheenumerationofCaspase3/7

positivecellsoverthetime

CeligoDemonstrationExperiment–HPCproliferationmeasurementusingKi-67 621001381 Rev A

Assay Name: HPC proliferationmeasurement using Ki-67 cellularmarkerAssayID:Celigo_02_0014


Experiment:HPCproliferationmeasurementusingKi-67cellularmarker

Purpose TodemonstratethecapabilityoftheCeligotoperformrapid,highthroughputimagingandanalysisofhematopoieticprogenitorcell(HPC)proliferationusingKi-67cellularmarker.TheKi-67proteinisabiomarkerforcellproliferationwhicharepresentinalltheactivephasesofcellcyclesuchasG1,S,G2,andmitosis,butnotinG0phase.

CurrentMethod(s) Flowcytometry,butistedioustosetupmultiplesamples,notideaforhigh-throughputassays

TargetCellType HPCsderivedfromiPSCs(onehealthyandonediseasedpatient)ExperimentPlan Cellswereculturedin6-wellplates,samplesfromdiseasedandhealthydonors,

fixed,permeabilized,andstainedforKi-67expression,andcounterstainedwithDAPI.

Hypothesis Celigowillbeabletoperformrapid,whole-wellimagingandanalysisofKi-67expressionlevelstocomparebetweengroups.

CeligoSetupPlateType 6-wellCorningScanChannels Brightfield,Green(DyLight488),Blue(DAPI)Resolution 1um/pixelScanArea WholewellAnalysisMethod Target1+2+MaskScanFrequency Once(endpoint)ScanTime 10minutes



Goal

TodemonstratethecapabilityoftheCeligotoperformrapid,highthroughputimagingandanalysisofcellproliferationusingKi-67cellularmarker.

Protocol

Cellandstainpreparation

1. CollecteddifferentsamplesofiPSCsthatwereisolatedfromeitherhealthyanddiseaseddonors2. HPCswereplatedinto6-wellplatesandincubatedfor2days(Seeplatemapbelow)

1 2 3A HealthyDonorSamplesB DiseasedDonorSamples

3. Atendofincubation,cellswerefixedwith4%formaldehyde,permeabilizedwith0.2%TritonX-1004. Thecellswerethenstainedwithprimaryrabbitanti-humanKi-67overnight5. Afterovernightstaining,thecellswerewashedandstainedwithsecondaryDyLight488goatanti-rabbit

IgGantibodyfor1hour6. Finally,theywerewashedandcounterstainedwithDAPIfor30mininthedark7. ThestainedcellswereimagedandanalyzedonCeligoforendpointreading

DataCollection

6. Immediatelyafter,theplatewasscannedinCeligousingTarget1(BF)+2(Green)+Mask(Blue)foranendpointscan

7. FluorescentgatingwassetupbasedontheDAPImasktodeterminemeanfluorescentintensityand%ofKi-67positivecells

DataAnalysis

• TheimagesforeachHPCsamplewereanalyzedbyusingtheDAPI-positivecellsasthemask• Next,theDyLight488fluorescentintensitieswithintheidentifiedDAPI-positivecellswereplottedunder

theGatetabtodeterminetheDyLight488-positivecellpopulationpercentages


Results1.Celigo-capturedbrightfield,DyLight288andDAPIfluorescentimages

• Examplesofbrightfield,Ki-67-DyLight488andDAPI-stainedfluorescentimages

• CeligowasabletocountDAPI-positivecells,todeterminethetotalpopulation,usethegatingfunctiontoidentifyKi-67-positivecellsandcalculatethepercentofKi-67-positivecellsforthewholeplate

BrightField Ki-67-DyLight488 DAPI


• CeligowasabletocalculatethepercentofKi-67-positivecellsfortheindividualwellsoftheentireplate

• Celigowasabletogeneratereportsintableformat

%Ki-67+ 1 2 3A 79.66% 82.16%B 76.73% 77.46%%Ki-67- 1 2 3A 20.29% 17.77%B 23.22% 22.47%

Conclusion• CeligowasabletoimageandidentifyKi-67-positivecellpopulationpercentageswiththeCeligo

gatingfunction• ThepercentofKi-67-positivecellpopulationwasautomaticallygeneratedbyCeligosoftware• Inthisexperiment,thecellproliferationfordiseasedpatientsampleswasnotsignificantlylower

thanthehealthypatientsshownintheKi-67cellpopulationpercentagesabove• CeligoimagecytometerallowedrapidbrightfieldandfluorescentimagingofHPCslabeledwith

DyLight488andDAPI

70.00%

75.00%

80.00%

85.00%

1 2CellPo

pulatio

n%

Ki-67CellPopulation

Healthy Diseased

CeligoDemonstrationExperiment–Antibody-DependentReceptorInternalizationAssay 671001390 Rev A

AssayName:Antibody-DependentReceptorInternalizationAssayAssayID:Celigo_02_0015


Experiment:Antibody-DependentReceptorInternalizationAssay

Purpose TomeasurethelevelofreceptorinternalizationinducedbyknownantibodyCurrentMethod(s) FlowCytometryTargetCellType GFPexpressingHT-293celllineExperimentPlan Comparereceptorinternalizationlevelbetweenapositivecontrolantibodyand

anegativecontrolantibodyatdifferentconcentrationsHypothesis Resultswillshowincreaseininternalizationcorrelatingtoincreasedfluorescent

signalsfromthepositivecontrolgroup.

CeligoSetupPlateType Greiner96-well,blackwallclearbottom,Cat#655090ScanChannels Brightfield,Red,GreenResolution 1µm/pixelScanArea WholewellAnalysisMethod Target1+2+MaskScanFrequency Hourly,upto5hoursScanTime ~9min



Goal

Tomeasurethelevelofreceptorinternalizationinducedbyknownantibody.

Protocol

Cellpreparation

20. Collectedtargetcellsandseededat10,000cells/wellintoeachwellusingtheplatemapbelowa. WellslabledwithMediaarecontrolwellswithcellsandmediaonlyb. N1-N5areserialdilutionofthenegativeantibody,whereN5isthehighestconcentrationc. P1-P5areserialdilutionofthepositiveantibody,whereP5isthehighestconcentration

21. Pipettedthepositiveandnegativeantibodiesatdifferentconcentrationsfollowingtheplatemapbelow22. Theantibodieswerepre-labeledwithapH-sensitivedyeusingakitfromPromegaandbindtoreceptors

onthetargetcells23. TheplateswerethenscannedusingtheCeligoatT=0,1,2,3,4and5hours

Drug 1 2 3 4 5 6 7 8 9 10 11 12

A

B Media N1 N2 N3 N4 N5

C Media P1 P2 P3 P4 P5

D Media N1 N2 N3 N4 N5

E Media P1 P2 P3 P4 P5

F

G

H

DataCollection

8. Afteraddingthecellsandantibodies,theplatewasscannedinCeligousingTarget1+2+Maskapplicationfort=0h

9. Repeatthescanningfort=1,2,3,4,and5h

DataAnalysis

• TheimagesateachtimepointwereanalyzedtocountthetotalnumberofGFPpositivecells• Next,theredfluorescentintensitywasmeasuredfromwithinthecellstodeterminethelevelof

receptorinternalizationcomparedtothenegativecontrols


Results1.ReceptorInternalizationimagesusingCeligoImagingCytometer

• Celigowasusedtocapturebrightfieldandfluorescentwhole-wellimageson96-wellplates• Itrequired~9min/plateforimageacquisitionandfluorescentdataanalysis• Wholeplateoverviewcanbeviewedtoquicklyassessthereceptorinternalizationresults(Seefigure

below)o Inthisexample,weshowedaplateviewofHT-293at5hourontheCeligosoftwaretoprovidea

quickat-a-glanceresultofthereceptorinternalizationo Thevaluesshownineachwellarethetotalorintegratedfluorescentintensities


• Eachwellcanbefurtherzoomed-intoviewimagesofindividualwellsandcellpopulationsinthewells,whichallowsvisualconfirmation(Seefigurebelow)

o Inthisexample,weshowedHT-293,zoomed-inviewat5houranddifferentAntibodytreatmentconcentrations

o Itisclearthattheredfluorescenceincreasedastheconcentrationofpositiveantibodyincreased


2.Antibody-dependentreceptorinternalizationresults• Thereceptorinternalizationanalysismeasuredthetotalfluorescentintensityinthecellpopulation

treatedwithdifferentAntibodyconcentrations• TheNegativeAntibodyshowednofluorescentsignals,whichwasobservedintheimagesaswell• Bymeasuringthetotalfluorescentintensitiesinthecells,wesawanincreaseinsignalasthe

concentrationofpositiveAntibodyincreased

• Inaddition,Celigosoftwarewasabletoperformbackgroundcorrectiontoimprovesignal-to-backgroundratiowithresultingdatashowingbelow

18000.019000.020000.021000.022000.023000.024000.025000.0

0.00 0.06 0.13 0.25 0.50

Integrated

Intensity

[Ab](µg/ml)

ReceptorInternalizationwithoutbackgroundcorrection

Negative Positive

0.0500.0

1000.01500.02000.02500.03000.03500.0

0.00 0.06 0.13 0.25 0.50 1.00

Integrated

Intensity

[Ab](µg/ml)

ReceptorInternalizationwithbackgroundcorrection

Negative Positive


Conclusion• Theresultsshowedacleardifferencebetweennegativeantibodyandpositiveantibodyin

internalization• Adoseresponsewasobservedforpositiveantibodybymeasuringaveragetotalfluorescentintensity• ByusingthebackgroundcorrectionfunctiononCeligo,thebackgroundcanberemovedtoincreasethe

fluorescentintensities

CeligoDemonstrationExperiment–Countandmeasurecellinfectivityofmicrocarriers 741001399 Rev A

AssayName:CountandmeasurecellinfectivityofmicrocarriersAssayID:Celigo_02_0016


Experiment:Countandmeasurecellinfectivityofmicrocarriers

Purpose Inthisexperiment,wedemonstratethecapabilityofCeligotomeasurecellcountonmicrocarrierbeadsandinfectedcellspositivelystainingforAlexaFluor488-labeledantibodyagaintheviralprotein.

CurrentMethod(s) FlowCytometryTargetCellType EpithelialcellsculturedonmicrocarriersExperimentPlan UsetheCeligotocountDAPI-stainedcellsonthemicrocarrierbeads,andcount

thenumberofmicrocarrierstogetanaveragecells/microcarrier.AlsomeasureAlexaFluor488-positivecellsonthemicrocarriers(cellsarestainedagainstviralprotein-forviralinfectivity)

Hypothesis Dependingontheinfectionrate,differentnumberofAlexaFluor488-positivecellswillbequantified,andaverageinfectivitywillbecalculated

CeligoSetupPlateType Greiner65509096-wellblackwallclearbottomScanChannels Brightfield+Green+BlueResolution 1µm/pixelScanArea WholewellAnalysisMethod Target1+2+3+4

Brightfield–ColonyforMicrocarriercountsScanFrequency EndpointScanTime ~10min



Goal

Inthisexperiment,wedemonstratethecapabilityofCeligotomeasurecellcountonmicrocarrierbeadsandinfectedcellspositivelystainingforAlexaFluor488-labeledantibodyagaintheviralprotein.

Protocol

Cellpreparation

• Obtainedmicrocarriersamplesfrombioreactors,fixedandstainedwithDAPIandviralproteinofinterestwithAF488

• Afterpipettinginthemicrocarrierbeadsfromspinnerflasksinto96-wellplates,centrifugedtheplatetosettlethemicrocarriersdown

• UsedtheCeligotoscanthemicrocarrierbeadsatdifferentfocalplanestocaptureallthenuclei• Inaddition,brightfieldimageswerecapturedforthemicrocarriers,tocountthenumberofmicrocarriers

inthewell• TheexperimentwasrepeatedbystainingwithHoechstandPItomeasuretheviabilityofepithelialcells

onthemicrocarrierso Notes:PotentiallystainingthecellswiththeCaspase3/7kitfollowingtheattachedprotocolto

measureapoptosis

DataCollection

10. Aftercentrifugingtheplate,scanedtheplateusingtheCeligo11. Setupthescanningparametersfor4channels,wherechannel1and2wereDAPIandAF488forthetop

ofthemicrocarriers,andchannel3and4wereDAPIandAF488forthebottomofthemicrocarriers12. TheCeligowasnotabletoimageandanalyzetheequatorofthemicrocarriers13. TheCeligowasthenusedtocapturebrightfieldimagesandanalyzethenumberofmicrocarriersinthe

well

DataAnalysis

• TheimagesfromeachDAPIandA488fluorescentchannelswerecounted• ThetotalnumberofcellsstainedwithDAPIwerecounted• ThetotalnumberofAF488-positivecellswerecounted• TheAF488#/DAPI#wascalculatedtodeterminetheInfectivity%• TheAf488#/BF#wascalculatedtodeterminetheaverageinfectedcells/microcarrier


Results1.Celigo-capturedbrightfieldandfluorescentimagesofDAPIandAlexaFluor488

• TheCeligowasabletomeasure%infectivitybycountingtotalnumberofDAPIandAlexaFluor488positivecells

• BydividingAlexaFluorbyDAPI,the%infectivitywascalculated


• Thewholewellimageshowedallthemicrocarriersinthewellandwerecounted

• Whenzoomedin,thecoverageofcellscanbeclearlyobservedintwodifferentsamples,B4andB12


2.Infectivitypercentageresults• AftermeasuringDAPIandAlexaFluor488-positivecells,aswellasthenumberofmicrocarriersper

sample,the%infectivityandaverageinfectedcells/beadwerecalculated• Theresultsshowedthatdifferentsampleshavedifferentrateofinfection

0%

5%

10%

15%

20%

25%

30%

1 2 3 4 5 6 7 8

Infectivity

%

Sample

Infectivity%

02468101214161820

1 2 3 4 5 6 7 8

Infected

cells/bead

Sample

AverageInfectedcells/bead


3.Celigo-capturedbrightfieldandfluorescentimagesofcellsandmicrocarriersforHoechstandPI• TheCeligowasusedtocapturebrightfieldimagesofthemicrocarriersforcounting• TheCeligowasalsousedtocaptureHoechstandPIstainedcellsgrowingonthemicrocarriersinorderto

measurethetotalcellcountsperwell,andpermicrocarrier• IndividualHoechstandPI-positivecellswerecounteddirectlyinthe96-wellplate,aswellasthe

microcarriers,shownintheimagesbelow.



• Thecellviabilitywasmeasureddirectlyonthemicrocarriers

Conclusion• Celigowasabletodirectlymeasuretotalcellcountandviralinfectedcellcountsofcellsonthe

microcarriersina96-wellplateformat• TheCeligowasalsousedtoquantifyvirallyinfectedcellsperbeadinahighthroughputmanner

o TheCeligoanalyzed20samplesinlessthan10minutestotalforscanningandanalysis• Celigowasalsoabletocaptureblueandredfluorescentimagestoperformtotalanddeadcell

countingusingHoechstandPIo Inaddition,thenumberofmicrocarrierswasalsocountedinthebrightfieldimageso Overall,theCeligowasabletocaptureandanalyze64samplesin15min

• Finally,highqualityimagescanbesavedandreviewedforrecordkeeping

0%10%20%30%40%50%60%70%80%90%100%

1 2 3 4 5 6 7 8

Viability(%

)

Sample

CellViability

CeligoDemonstrationExperiment–GFPTransfectionEfficiencyMeasurement 831001403 Rev A

AssayName:GFPTransfectionEfficiencyMeasurementAssayID:Celigo_02_0018


Experiment:GFPTransfectionEfficiencyMeasurement

Purpose Toanalyzetransfectionefficiencyovera4dayperiodwhentreatedwithvariousamountsofatransfectioncompound.

CurrentMethod(s) ManualobservationusingfluorescentmicroscopeTargetCellType 293HcellsExperimentPlan Platetransfectedcellsandmeasuretransfectionefficiencyfor4daysHypothesis HigherviraldosagetreatmentswillleadtohigherGFPnumbersandfluorescent

intensities

CeligoSetupPlateType Greiner65509096-wellblackwallclearbottomScanChannels Brightfield+GreenResolution 1µm/pixelScanArea WholewellAnalysisMethod ConfluenceRatio:Confluence1+2ScanFrequency DailyScanTime ~8min



Goal

Toanalyzetransfectionefficiencyovera4dayperiodwhentreatedwithvariousamountsofatransfectioncompound.

Protocol

Cellpreparation

• TransfectedcellswerecollectedandplatedonDay0followingtheplatemapbelow

1 2 3 4 5 6 7 8 9 10 11 12A B

60,000cells/well 80,000cells/well

C D E F G

• Afterplating,theplatewascentrifugedtosettlethecellsinthebottomofthedish,inordertoimagethecellsinamonolayerforoptimalfocus

• Cellswerethentreatedwithcompoundsfollowingtheplatemapbelow

1 2 3 4 5 6 7 8 9 10 11 12A B Control Low High Low High High Low High Low Control C D E F G

• TheplatewasthenimagedusingCeligoforbrightfieldandGFPgreenfluorescence• TheplatewasimagedandanalyzedonDay0,1,2,and3


DataCollection

14. Aftercentrifugingtheplate,theplatewasscannedusingtheCeligo15. Thescanningparametersfor2channelsweresetup,whereConfluencechannel1+2areGFPand

brightfield,respectively

DataAnalysis

• Theimageswereanalyzedtomeasuretotalareaofcellcoverageinbrightfieldandinfluorescence• TheautomaticallycalculatedconfluenceratioindicatesGFPtransfectionefficiencyasapercentageof

thetotalcellarea

Results1.GFPexpressionfluorescentimages

• TheGFPfluorescentimagesshoweddifferencesbetweenlowandhighcompoundtreatment• TheGFPexpressionplateviewisshownbelow


2.GFPtransfectionefficiencymeasurementresults• Whole-wellfluorescentimagesrevealtheGFPexpressionlevelforcellstreatedwithhighandlow

concentrationofcompound

• TheGFPtransfectionpercentagefor60,000cells/wellshowednoexpressionforthenegativecontrol,lowcompoundtreatmentshowed60%,andthehighcompoundtreatmentshowed100%

• Theresultsweresimilarforthe80,000cells/well

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0 0.5 1 1.5 2 2.5 3 3.5

GFPPo

pulatio

n%(C

onflu

enceRatio)

Time(day)

6x10^4cells/well

Control

HighCompound

LowCompound

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0 0.5 1 1.5 2 2.5 3 3.5

GFPPo

pulatio

n%(C

onflu

enceRatio)

Time(day)

8x10^4cells/well

Control

HighCompound

LowCompound


3.Brightfieldconfluencelevel• Thebrightfieldimagesshowedthatathighcompoundtreatment,thereisreductionincellconfluency,

incomparisontothelowcompoundtreatment


• At60,000cells/well,thehighcompoundtreatmentshowedalargedecreaseincellconfluency• At80,000cells/well,thehighcompoundtreatmentdidnotshowanydecreaseincellconfluency

4.GFPtransfectiontime-courseimages• GreenfluorescentimagesshowedanincreaseinthenumberofGFP-positivecellsover3daysofculture• ThelowcompoundtreatmentresultedinfewerGFP-expressingcellscomparedtothehighcompound

treatment

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0 0.5 1 1.5 2 2.5 3 3.5

Bright-FieldCon

fluence%

Time(day)

6x10^4cells/well

Control

HighCompound

LowCompound 0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0 0.5 1 1.5 2 2.5 3 3.5

Bright-FieldCon

fluence%

Time(day)

8x10^4cells/well

Control

HighCompound

LowCompound


Conclusion• TheGFPtransfectionefficiencieswerehighlydependentonthecompoundtreatment• Atlowerseedingdensity,thecellsshowedhigherdamageduetohighcompoundtreatment

CeligoDemonstrationExperiment–Antibody-DependantDrugUptakeAssay 911001404 Rev A

AssayName:Antibody-DependentDrugUptakeAssayAssayID:Celigo_02_0019


Experiment:Antibody-DependentDrugUptakeAssay

Purpose Measurecellulardruguptakeinadherentandsuspensioncellculturesofafluorescently-labeleddrugatvaryingantibodyconcentrations

CurrentMethod(s) FlowCytometryTargetCellType HumannormalskinfibroblastExperimentPlan Targetcellsareplated,thenAF488-drugandantibodyareadded.UsetheCeligo

toscantheplateusingbrightfieldandgreenfluorescentchannelsHypothesis Theamountofdruguptakechangesaredependentonantibodyanddrug

concentrations

CeligoSetupPlateType Greiner65509096-wellblackwallclearbottomScanChannels BrightfieldandGreenResolution 1µm/pixelScanArea WholewellAnalysisMethod Target1+MaskScanFrequency OnceScanTime ~15minutes



Goal

Thegoalofthisexperimentistomeasurecellularuptakeofafluorescently-labeleddrugandvaryingantibodyconcentrationsinanadherentandsuspensioncellculture.

Protocol

Suspensioncellpreparation

24. Skinfibroblastsweretrypsinizedandpipettedintoa96-wellplateat15,000cells/wellin200µlofmedia,followingtheplatemapbelow

25. Thecellswereincubatedovernight26. Afterincubation,differentconcentrationsofAlexaFluor488-Drugandantibodieswereaddedtothe

wellsandincubatedfor3hours(PlateMapBelow)27. Afterincubation,thecellsweretrypsinizedandwashed28. Thecellswerereseededat200µlinPBSintoanew96-wellplate29. TheplatewasimagedandanalyzedusingCeligo

D2 D1

1 2 3 4 5 6 7 8 9 10 11 12

Ab20 A 15K 15K 15K 15K

Ab15 B 15K 15K 15K 15K

Ab10 C 15K 15K 15K 15K

Ab5 D 15K 15K 15K 15K

Ab2.5 E 15K 15K 15K 15K

Ab1 F 15K 15K 15K 15K

Ab0.5 G 15K 15K 15K 15K

Ab0 H 15K 15K 15K 15K


Adherentcellpreparation

1. Skinfibroblastsweretrypsinizedandpipettedintoa96-wellplateat15,000cells/wellin200µlofmedia,followingtheplatemapbelow

2. Thecellswereincubatedovernight3. Afterincubation,2differentconcentrationsofAlexaFluor488-Drugandantibodieswereaddedtothe

wellsandincubatedfor3hours(PlateMapBelow)4. Afterincubation,themediawasreplacedwith200µlofPBS5. TheplatewasimagedandanalyzedusingCeligo

Control D2 D1 D0.5

1 2 3 4 5 6 7 8 9 10 11 12

Ab20 A 15K 15K 15K 15K 15K 15K

Ab15 B 15K 15K 15K 15K 15K 15K

Ab10 C 15K 15K 15K 15K 15K 15K

Ab5 D 15K 15K 15K 15K 15K 15K

Ab2.5 E 15K 15K 15K 15K 15K 15K 15K 15K

Ab1 F 15K 15K 15K 15K 15K 15K 15K 15K

Ab0.5 G 15K 15K 15K 15K 15K 15K 15K 15K

Ab0 H 15K 15K 15K 15K 15K 15K 15K 15K

DataCollection

16. TheplatewasscannedinCeligousingTarget1+2foranendpointreadinga. Target1isthegreenfluorescentchannelandTarget2isthebrightfieldchannel

DataAnalysis

• Theimagesforeachdrugandantibodyconcentrationwereanalyzedtocounttotalnumbercellsinthebrightfieldimages

• Next,thefluorescentintensitieswereplottedinahistogramundertheGateTabintheCeligosoftware• Finally,thepercentageofAlexaFluor488-positivecellsweremeasuredtodeterminetheeffectof

antibodyconcentrationsondruguptakelevel


Results1.Celigogatingfunctionforfluorescentintensityanalysis

• Cellimageswereanalyzedbycountingthetotalnumberofcells(Hoescht)andcellsweregatedbasedonthemeanfluorescentintensitytodeterminepercentofdruguptake

Control,untreated(AF488+Hoescht)

Drug2,Ab0.5(AF488+Hoescht)

Drug2,Ab20(AF488+Hoescht)


2.Celigo-capturedAlexaFluor488fluorescentimagesforsuspensioncells

• Brightfieldandfluorescentimagesatdifferentdrugandantibodyconcentrationsforsuspensioncells• RepresentativeimagesofcellsincubatedwithDrug2and1atahigh(20)andlow(0.5)antibody

concentration


3.PercentcellularuptakeofAlexaFluor488-drugforsuspensioncells• Totalcellswerecountedusingthebrightfieldchannelandthemeanfluorescentintensitieswere

measuredwithineverycountedcell• Lowerantibodyconcentrationsincubatedwiththefluorescently-labeleddrugresultedinhigher

percentageofuptakeofthedrugintothecells• Inthewellswithhigherantibodyconcentrations,drugsappearedtobeaggregatingextracellularlyand

wereexcludedfromthefluorescentintensitydata

0%10%20%30%40%50%60%70%80%90%

100%

Ab20 Ab15 Ab10 Ab5 Ab2.5 Ab1 Ab0.5 Ab0

%uptakeofAF488-drug

Antibodyconcentration

Antibody-DependentDrugUptakeAssayTrypsinized

Drug2

Drug1


4.Celigo-capturedAlexaFluor488fluorescentimagesforadherentcells

• BrightfieldandfluorescentimagesatatdifferentDrugandAntibodyconcentrationsforadherentcells• RepresentativeimagesofcellsincubatedwithDrug2,1,and0.5atahigh(20)andlow(0.5)antibody

concentration


CeligoDemonstrationExperiment–Antibody-DependantDrugUptakeAssay1001001404 Rev A

5.PercentcellularuptakeofAlexaFluor488-drugforadherentcells• TotalcellsweredeterminedbyHoeschtstainingandbrightfieldimageswerecapturedfor

morphologicalobservation• MeanAlexaFluor488fluorescentintensitiesweremeasuredfromtheareasurroundingthenucleifor

everycellcounted

Conclusion• Adherentculturesofhumanskinfibroblastswerepreparedforadruguptakeassay• Uptakeofthefluorescentdrugwasinhibitedatthehigherantibodyconcentrationsinbothsuspension

andadherentcultures• Usingadherentcellsinthemicroplateformatallowedresearcherstoobservethemorphological

changesinthecellculture• Theresultsshowedanincreaseinpercentuptakeasdrugconcentrationincreased• Incontrast,thepercentageuptakeincreasedastheantibodyconcentrationdecreased• Scanningofone96-wellplateinthreechannels(brightfield,greenfluorescence,bluefluorescence)

requiredlessthan15minutes

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Ab20 Ab15 Ab10 Ab5 Ab2.5 Ab1 Ab0.5 Ab0

%uptakeofAF488-drug

Antibodyconcentration

Antibody-DependentDrugUptakeAssayAdherent

Drug2 Drug1 Drug0.5

CeligoDemonstrationExperiment–Label-freeTumorSpheroidGrowthInhibition1011001383 Rev A

AssayName:Label-freeTumorSpheroidGrowthInhibitionAssayID:Celigo_03_0001


Experiment:Label-freeTumorSpheroidGrowthInhibition

Purpose Toprovideanautomatedsolutionthatcanprovideimagesofmulticellulartumorspheroids(MCTS)andreportMCTSdiameterina96-wellformat

CurrentMethod(s) MicroscopyTargetCellType NCI-H460,MiaPacaExperimentPlan Scantwoplatesdailytomeasurethesizeofmulticellulartumorspheroidsunder

differentdrugconcemtrationsinhypoxicornormoxicconditionsHypothesis Usingthebrightfieldimaging,theCeligowillrapidlyprovidemulticellulartumor

spheroidimagesanddiametersoftreatedMCTSina96-wellplate

CeligoSetupPlateType Corning™CLS70007(ULAplate)-96wellplateScanChannels BrightfieldResolution 3micron/pixelScanArea WholewellAnalysisMethod Tumorsphere1ScanFrequency DailyScanTime 3minutes



Goal:

Imageandmeasurethediameterofmulticellulartumorspheroids(MTSs)atdifferentdrugconcentrationsthatwereincubatedateithernormoxicorhypoxicconditions.Ultimatelydeterimine,ifincubationconditions(normoxic,andhypoxic)playaroleinthereductionofMTSsizewhentreatedatdifferentdrugconcentrations.

ProtocolCellPreparation

1. Seeded2,500NCI-H460cells/wellinaULA96-wellplates(seeplatemapbelow)2. Seeded1,250MiaPacacells/wellinaULA96-wellplates(seeplatemapbelow)3. Onday4,treatedformedMTSswithdrugXorvehiclecontrol

a. DrugXin[µM]wasseriallydiluted1/3from10µMto0.0045µMconcentration4. Placedoneplateinhypoxicconditionsandoneplateatstandardgrowthconditions

Platemap

AplateatNormoxiaandHypoxia

DataCollection

17. Onday4,afteraddingthedrugatdifferentconcentrations,theplateswereimagedanddatacollectedforboththehypoxiaandnormoxiaplates

18. Theplateswereagainimaged4dayslater.Posttreatment,oneplatewasathypoxicconditionsfor4daysandthesecondplatewasnormoxicconditions

19. TheplateswaerescannedinCeligousingTumorsphere1(brightfield)assay

NCI-H460

2,500Cells/well

MiaPaca

1,250Cells/well

CC4.5nM1/31/31/31/31/31/310µM

CC4.5nM1/31/31/31/31/31/310µM

CC4.5nM1/31/31/31/31/31/310µM

CC4.5nM1/31/31/31/31/31/310µM

CC4.5nM1/31/31/31/31/31/310µM

CC4.5nM1/31/31/31/31/31/310µM

4.5nM 10µM(1/3)SerialDilution


DataAnalysis

• TheimageswereanalyzedbyusingTumorsphere1applicationtoidentifytheMCTSinthewell• Thewellmaskwasreducedtothe95thpercentiletoeliminateedgeeffects• Severalwellshadmultiplemulticellulartumorspheroids.Inthoseinstances,eachspheroidwas

identifiedbytheCeligosoftware

Results

1.NCI-H460multicellulartumorspheroidsshowedanoticeabledecreaseinspheroidsizeathighdrugconceration

• NCI-H460multicellulartumorspheroidsshowedadecreaseinspheroidsizefrom780µmat3µMdrugconcentrationto750µmatnormoxicand720µmathypoxicconditionsat10µM.

• MiaPacamulticellulartumorspheroidsshowednochangeinspheroiddiameterbetweenhypoxicandnormoxicconditionsforcontrolordrugtreatedsamples.

• ImageandgrapheddatabelowrepresentsNCI-H460andMiaPacamulticellulartumorspheroidstreatedwithDrugXinadosedependedntmannerandincubatedatnormoxicandhypoxicenvironmbetforfourdaysbeforeimagingontheCelgio.

Day4posttreatmentwithDrugXatNormoxiaNCI-H640at2,500andMiaPacaat1,250cells/well

• Theentireplatewasscannedin3minutesandbrightfieldthumbnailsareshownforeachwell.• AtthebottomofthewellpictureistheCeligomeasureddiameterin(microns)foreachidentified

spheroid.

NCI-H460

2,500Cells/well

MiaPaca

1,250Cells/well


Day4posttreatmentwithDrugXatHypoxiaNCI-H640at2,500andMiaPacaat1,250cells/well

• Theentireplatewasscannedin3minutesandbrightfieldthumbnailsareshownforeachwell.• AtthebottomofthewellpictureistheCeligomeasureddiameterin(microns)foreachidentified

spheroid.

Celigocapturedrepresentativebrightfieldimagesofnon-drugtreatedNCI-H460andMiaPacamulticellulartumorspheroidsatnormoxicconditiononday0

MiaPaca NCI-H460

NCI-H4602,500Cells/w

ellMiaPaca

1,250Cells/well


NCI-H460andMiaPacamulticellulartumorspheroidmeasurementsfourdaysafterdrugtreatmentandincubationatnormoxicandhypoxicconditions

• NCI-H460andMiaPacawereplatedat2,500and1,250cellsperwelltreatedwithaseriallydilutedDrugXandincubatedfor4daysateitherhypoxicornormoxicconditions

• NCI-H460multicellulartumorspheroidsshowedanoticeabledecreaseinspheroidsizeat10µMdrugconcentrationatbothnormoxicandhypoxicconditions.

• ThesizesofMiaPacamulticellulartumorspheroidsremainedthesamethroughttheexperimentatbothnormoxicandhypoxicconditionsaswellasatdifferentdrugconcentrations.

Conclusion• Usingthe96-wellU-bottomULAplates,wesuccessfullycapturedimagesofMCTSandanalyzedthedata

usingtheCeligoinstrument.• Theentire96-wellplatewasimagedin3minutes.Theshortscantimesignificantlyincreasesthe

throughputduringanexperimentthathasmultipleplates.• After4daysofdrugtreatmentandincubationateithernormoxicorhypoxicconditions,spheroid

diametersweremeasuredandautomaticallyreportedbytheCeligosoftware.Noadditionalsoftwareisrequiredforimageprocessingofmulticellulartumorspheroiddiameters

CeligoDemonstrationExperiment–3DMCTSinvasionscreeningassay1071001391 Rev A

AssayName:3Dmulticellulartumorsphere(MCTS)invasionscreeningassayAssayID:Celigo_03_0002


Experiment:3Dmulticellulartumorsphere(MCTS)invasionscreeningassay

Purpose MonitortheeffectsofapanelofdrugsontheinvasionofU87MGGlioblastomaMCTSintoBasementMembraneExtract(BME)Matrigel

CurrentMethod(s) MicroscopyTargetCellType U87MGExperimentPlan AllowU87MGspheroidstoformandtreatwithapanelofdrugcompounds,then

imageat0,17,23,41,47,and68hoursaftertreatmenttomeasurementtheinvasioninhibitioneffectsofthecompounds

Hypothesis Usingthebrightfieldimaging,theCeligowillrapidlyprovidemulticellulartumorspheroidinvasionimagesandtheareaoftheinvasionoftreatedU87MGMCTS

CeligoSetupPlateType NexcelomU-bottomUltra-lowAttachment384-wellPlate(Cat#ULA-384U)ScanChannels BrightfieldResolution 1µm/pixelScanArea WholewellAnalysisMethod TumorsphereMigrationScanFrequency 0,17,23,41,47,and68hoursScanTime ~4min



Goal:

ImageandanalyzetheinhibitoryeffectsofapanelofdrugcompoundsonU87MGMCTSinvasionintoBasementMembraneExtractovertime.


5. Seeded500U87MGcells/wellinULA384-wellplates6. Onday4,addedseriallydiluteddifferentdrugcompoundsat2xandavehiclecontrolinBasement

MembraneExtract(BME)Matrigel7. Monitoredinvasionbyimagingandanalyzingeach384-wellplateat~4min/plateat0,17,23,41,47,

and68hoursontheCeligoimagingcytometer8. Measuredtheinvasionarea0,17,23,41,47,and68hoursforeachdrugcompoundtreatedMCTS9. Comparedtheinvasionareaforeachdrugcompoundateachtimepointtocharacterizethetested

compounds

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 18 20 21 22 23 24

A

B

C

CNTL Cmpd1 Cmpd2 Cmpd3 Cmpd4 Cmpd5 Cmpd6 Cmpd7

5µM

D 2.5µM

E 1.25µM

F 0.625µM

G 0.3125µM

H

I J

CNTL Cmpd8 Cmpd9 Cmpd10 Cmpd11 Cmpd12 Cmpd13 Cmpd14

5µMK 2.5µML 1.25µMM 0.625µMN 0.3125µM

O

P


DataCollection

20. Afteraddingthedrugatdifferentconcentrationsonday4,theplateswereimagedanddatacollectedfortheentire384-wellplate

21. Theplateswereagainimaged0,17,23,41,47,and68hourspost-treatmentinordertoperformtimecoursemonitoringoftumorspheroidinvasionarea

DataAnalysis

• TheimageswereanalyzedbyusingtheTumorsphereMigrationapplicationtomeasuretheinvasionareaofMCTSinthewell

• TheinvasionareaofeachMCTStreatedwithdifferentdrugcompoundswasmeasured

Results

1.Time-coursebrightfieldimagesandresultsofMCTSforcontrolandtreatedsample• ThebrightfieldimagesshowedthecontrolMCTSincreasedininvasionareaover,timewhilethetreated

samplewithcompound8showedinvasioninhibition

• Thetime-courseresultsshowedthatsomedrugsinhibitedtheinvasioninthebeginningofthetreatment,andsomedrugsinhibitedseveraldaysafterthetreatment

• Somedrugcompoundsdidnotinduceinhibition,similartothecontrol


2.Dose-responsebrightfieldimagesandresultsofMCTSforcontrolandtreatedsample• Thebrightfieldimagesshowedthedoseresponseofcompound8invasioninhibitionofMCTS• Astheconcentrationincreased,theinvasionareadecreased

• Thedose-responseresultsshowedthatsomedrugsgeneratedgreatdoseresponse,somedrugsinhibitedinvasionateveryconcentration,andsomedrugdidnothaveanyeffect

0100000200000300000400000500000600000700000800000

0 20 40 60 80

Invasio

nArea(µ

m2)

Time(hour)

Time-CourseMonitoringofSpheroidInvasionAreaControl1

2

3

4

5

6


3.EndpointresultsofMCTSforcontrolandtreatedsample• Theendpointresultsshowedthatdrugcompounds2,3,4,5,7,8,11,13,and14inhibitedtheinvasion

ofU87MGMCTS• Drugcompounds1,6,9,10,and12didnotinhibitMCTSinvasion

0100000200000300000400000500000600000700000800000900000

0.1 1 10

Invasio

nArea(µ

m2)

[Compound](µM)

Dose-responseEffectonSpheroidInvasionArea1

2

3

4

5

6

7

8

0

200000

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800000

1000000

CNTL 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Invasio

nArea(µ

m2)

Compounds

AverageInvasionAreaat5µMofDrug


Conclusion• Usingthe384-wellU-bottomULAplates,wesuccessfullycapturedimagesofU87MGMCTSand

analyzedthedatausingtheCeligoimagingcytometer• Theentire384-wellplatewasimagedin~4min.Theshortscantimesignificantlyincreasesthe

throughputduringanexperimentthathasmultipleplates• Afterthedrugtreatments,spheroidinvasionareasweremeasuredandautomaticallyreportedbythe

Celigosoftware• NoadditionalsoftwarewasrequiredforimageanalysisofMCTSdiametersinvasionarea

CeligoDemonstrationExperiment–3DMCTSgrowthinhibitionscreeningassay1141001393 Rev A

AssayName:3Dmulticellulartumorspheroid(MCTS)growthinhibitionscreeningassayAssayID:Celigo_03_0004


Experiment:3Dmulticellulartumorspheroid(MCTS)growthinhibitionscreeningassay

Purpose MonitortheeffectsofapanelofdrugsonthegrowthinhibitionofU87MGGlioblastomaMCTS


imageon0,69,114,165,210hourspost-treatmenttomeasurementthegrowthinhibitioneffectsofthecompounds

Hypothesis Usingthebrightfieldimaging,theCeligowillrapidlyprovidemulticellulartumorspheroidimagesanddiametersoftreatedU87MGMCTS

CeligoSetupPlateType NexcelomU-bottomUltra-lowAttachment384-wellPlate(Cat#ULA-384U)ScanChannels BrightfieldResolution 1µm/pixelScanArea WholewellAnalysisMethod Tumorsphere1ScanFrequency 0,69,114,165,210hourspost-treatmentScanTime ~4min


AssayProtocolandPlateSetupGoal:

ImageandanalyzethegrowthinhibitioneffectofapanelofdrugcompoundsonU87MGMCTSovertime.


10. Seeded500U87MGcells/wellinULA384-wellplates11. Onday4,addedseriallydiluteddifferentdrugcompoundsat2xandavehiclecontrolinmedia12. Monitoredgrowthinhibitionbyimagingandanalyzingeach384-wellplateat~4min/plateon0,69,114,

165,210hourspost-treatmentwiththeCeligoimagingcytometer13. Measuredthespheroiddiameterson0,69,114,165,210hourspost-treatmentforeachdrug

compoundtreatedMCTS14. Comparedthespheroiddiametersforeachdrugcompoundateachtimepointtocharacterizethetested

compounds

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 18 20 21 22 23 24

A

B 10µM

Cmpd1 Cmpd2 Cmpd3 Cmpd4 Cmpd5 Cmpd6 Cmpd7

CNTL

C 5µM

D 2.5µM

E 1µM

F 0.5µM

G 0.1µM

H 0.05µM

I 10µM


J 5µM

K 2.5µM

L 1µM

M 0.5µM

N 0.1µM

O 0.05µM

P


DataCollection

22. Afteraddingthedrugatdifferentconcentrationsonday4,theplateswereimagedanddatacollectedfortheentire384-wellplate

23. Theplateswereagainimagedon0,69,114,165,210hourspost-treatmentinordertoperformtimecoursemonitoringoftumorspheroiddiameter

DataAnalysis

• TheimageswereanalyzedbyusingtheTumorsphere1applicationtoidentifytheMCTSinthewell• ThediameterofeachMCTStreatedwithdifferentdrugcompoundswasmeasured

Results

1.Time-coursebrightfieldimagesandresultsofMCTSforcontrolandtreatedsample• ThebrightfieldimagesshowedthecontrolMCTSincreasedindiameterovertimewhilethetreated

samplewithcompound8showedgrowthinhibition


• Thetime-courseresultsshowedthatsomedrugsinhibitedthegrowthinthebeginningofthetreatment,andsomedrugsinhibitedseveraldaysafterthetreatment

• Somedrugcompoundsdidnotinduceinhibition,similartothecontrol

2.Dose-responsebrightfieldimagesandresultsofMCTSforcontrolandtreatedsample• Thebrightfieldimagesshowedthedoseresponseofcompound8growthinhibitionofMCTS• Astheconcentrationincreased,thespheroidsizedecreased

200

250

300

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450

500

550

600

650

0 50 100 150 200 250

Sphe

roidDiameter(µ

m)

Time(hour)

Time-CourseMonitoringofSpheroidDiameter Control1234567891011121314


• Thedose-responseresultsshowedthatsomedrugsgeneratedgreatdoseresponse,somedrugsinhibitedgrowthateveryconcentration,andsomedrugdidnothaveanyeffect

3.EndpointresultsofMCTSforcontrolandtreatedsample• Theendpointresultsshowedthatdrugcompounds2,3,4,5,7,8,11,12,13,and14inhibitedthe

growthofU87MGMCTS• Drugcompounds1,6,9,and10didnotinhibitgrowth

0

100

200

300

400

500

600

700

0.01 0.1 1 10 100

Sphe

roidDiameter(µ

m)

[Compound](µM)

Dose-responseEffectonSpheroidDiameter1234567891011121314

050

100150200250300350400450500550600650700

CNTL 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Sphe

roidDiameter(µ

m)

Compounds

AverageSpheroidDiameterat5µMofDrug



analyzedthedatausingtheCeligoimagingcytometer• Theentire384-wellplatewasimagedin~4min.Theshortscantimesignificantlyincreasesthe

throughputduringanexperimentthathasmultipleplates• Afterthedrugtreatments,spheroiddiametersweremeasuredandautomaticallyreportedbytheCeligo

software• NoadditionalsoftwarewasrequiredforimageanalysisofMCTSdiameters

CeligoDemonstrationExperiment–3DMCTSendpointapoptosisscreening1211001397 Rev A

AssayName:3Dmulticellular tumor spheroidendpoint apoptosisscreeningAssayID:Celigo_03_0005


Experiment:3Dmulticellulartumorspheroid(MCTS)endpointapoptosisscreeningassay

Purpose MonitortheeffectsofapanelofdrugsontheapoptosisofU87MGGlioblastomaMCTSusingCaspase3/7andHoechstfluorescentstaining


imageonday13tomeasuretheapoptoticeffectsofthecompoundsHypothesis Usingthebrightfieldandfluorescentimaging,theCeligowillrapidlyprovide

multicellulartumorspheroidimages,andmeasureCaspase3/7andHoechstfluorescentintensitiesoftreatedU87MGMCTS

CeligoSetupPlateType NexcelomU-bottomUltra-lowAttachment384-wellPlate(Cat#ULA-384U)ScanChannels Green,Blue,andBrightFieldResolution 1µm/pixelScanArea WholewellAnalysisMethod Tumorsphere1+2+MaskScanFrequency EndpointScanTime ~8min



ImageandanalyzetheapoptoticeffectsofapanelofdrugcompoundsonU87MGMCTSonday13.


15. Seeded500U87MGcells/wellinULA384-wellplates16. Onday4,addeddifferentseriallydiluteddrugcompoundsat2xandavehiclecontrolinmedia17. Onday13,preparedandaddedCaspase3/7andHoechstforstainingtheMCTS18. Incubatedtheplateat37°Cand5%CO2for60min19. ImagedandanalyzedonCeligo20. ComparedthespheroidCaspase3/7fluorescentintensityforeachdrugcompoundateachtimepointto

characterizethetestedcompounds

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 18 20 21 22 23 24

A

B 10µM


CNTL

C 5µM

D 2.5µM

E 1µM

F 0.5µM

G 0.1µM

H 0.05µM

I 10µM


J 5µM

K 2.5µM

L 1µM

M 0.5µM

N 0.1µM

O 0.05µM

P


DataCollection

24. Afterincubatingthespheroidswithdrugsatdifferentconcentrations,thespheroidswerestainedwithCaspase3/7andHoechst

25. Theplatewithstainedspheroidswasimagedonday1326. ThecapturedimageswerethenanalyzedintheCeligosoftwarefortheentire384-wellplate

DataAnalysis

• TheimageswereanalyzedbyusingTumorsphere1+2+MaskapplicationtoidentifytheMCTSinthewell

• ThefluorescentintensitiesofCaspase3/7weremeasuredforeachdrug-treatedMCTSorcontrol

Results

1.EndpointbrightfieldandfluorescentimagesandresultsofMCTSforcontrolandtreatedsample• Thebrightfieldimageswereusedtoidentifythespheroidsineachwell• TheCaspase3/7fluorescentintensitiesweremeasuredfromtheimages


• TheplotbelowisshowingtheCaspase3/7fluorescentintensities,whichindicatedtheapoptosisofeachMCTStreatedwiththedifferentdrugcompounds

• Only2drugcompoundsinducednoticeableapoptosisontheU87MGMCTS,whileotherdrugshadnoeffects


analyzedthedatausingtheCeligoimagecytometer• Theentire384-wellplatewasimagedin~8min.Theshortscantimesignificantlyincreasedthe

throughputduringanexperimentthathasmultipleplates• Afterthedrugtreatments,thespheroidCaspase3/7fluorescentintensitiesweremeasuredand

automaticallyreportedbytheCeligosoftware• NoadditionalsoftwarewasrequiredforimageanalysisofMCTSfluorescentintensities

0102030405060708090

100110120130140

CNTL 1 2 3 4 5 6 7 8 9 10 11 12 13 14

AVECa

spase3/7INT(R.U.)

Compounds

AverageCaspase3/7FluorescentIntensityat5µMofDrug

CeligoDemonstrationExperiment–Countingofpatient-derivediDCorganoids1261001400 Rev A

AssayName:CountingofPatient-DerivedIDCOrganoidsAssayID:Celigo_03_0006


Experiment:CountingofPatient-DerivedIDCOrganoids

Purpose Toimageandcountwholewellpopulationsoforganoidsinarapidmannerwithouthavingtotakemultiple,timeconsumingZ-stackedimages

CurrentMethod(s) Manually,orConfocalMicroscopyTargetCellType Patient-derivedintestinaldifferentiatedcells(IDC)organoidsExperimentPlan Organoidsarecultivatedin6-wellplatesembeddedinmatrigelandimagedin

brightfieldHypothesis Celigowillbeabletoperformrapid,whole-wellimagingandanalysisof

organoidsinahighthroughputmanner

CeligoSetupPlateType Corning12-wellmicroplateScanChannels BrightfieldResolution 1µm/pixelScanArea WholewellAnalysisMethod Tumorsphere1ScanFrequency OnceScanTime ~8min


AssayProtocolandPlateSetupGoal

Toimageandcountwholewellpopulationsoforganoidsinarapidmannerwithouthavingtotakemultiple,timeconsumingZ-stackedimages

Protocol

IDCorganoidspreparation

30. IDCorganoidswerecultivatedin12-wellmicroplatesandembeddedinmatrigelfollowingtheplatemapbelow

31. Next,theorganoidsweregrownfor20daysfromIDCprogenitorcellstoformtheorganoids32. TheorganoidswerealsotreatedwithH2O2attime=0,toinhibitthegrowthoftheorganoids

1 2 3 4A Control H2O2 B Control H2O2 C Control Matrigelonly,nocells

DataCollection

27. Theorganoidsimageswerecapturedusingbrightfieldimaginga. OneoftheControlwellswasusedtofocustheorganoidstosetthefocusZ-locationb. TheCeligoscanrequiredapproximately5min

DataAnalysis

• TheorganoidswerecountedusingCeligoapplicationTumorsphere1• TheIDCorganoidscountsweregenerated,aswellasthesizeoftheorganoids


Results1.Celigo-capturedbrightfieldimagesoforganoids

• Celigocapturedbrightfieldimagesin12-wellplatescontainingorganoidsinmatrigel• Belowisanexamplewholeplateimageofthecaptureddata

• Celigocapturedhighresolutionimagesinbrightfieldthatcanbezoomed-inonfromwholewell(below)


• TheCeligosoftwareaccuratelycountedorganoidsanddeclusteredthemintoindividualorganoids

2.PDOCountingResults• Celigowasusedtocountalltheorganoidsinthe12-wellplate• Thecountedresultsandthesizeanalysisoforganoidsareshownbelow

TumorsphereCount 1 2

A 27 80

B 123 36

C 48 0

AVGDiameter(µm) 1 2

A 282.5625 311.4015

B 305.0027 261.4917

C 290.1047 NaN

Conclusion• Celigowasabletocountthenumberoforganoidsdirectlyinthewells,andmeasuredthediameter

ofeachorganoid• Overall,therewasnocleardifferencebetweenthecontrolandH2O2treatedsamples• Celigosoftwarewasabletodeclusterorganoidsincloseproximity,toimprovethecountingaccuracy

ofthecurrentmethod

ZoomedinImage ZoomedinFilledImage

CeligoDemonstrationExperiment–3DMCTSendpointviabilityscreeningassay1311001406 Rev A

Assay Name: 3D Multicellular tumor spheroid (MCTS) endpointviabilityscreeningassayAssayID:Celigo_03_0007


Experiment:3Dmulticellulartumorspheroid(MCTS)endpointviabilityscreeningassay

Purpose MeasuretheeffectsofapanelofdrugsontheviabilityofU87MGGlioblastomaMCTSusingcalceinAMandPropidiumIodidefluorescentstaining

CurrentMethod(s) MicroscopyTargetCellType U87MG(Glioblastoma)ExperimentPlan AllowU87MGspheroidstoformandtreatwithapanelofdrugcompounds,then

imageonday13tomeasurethecytotoxicityeffectsofthecompoundsHypothesis Usingthebrightfieldandfluorescentimaging,theCeligowillrapidlyprovide

multicellulartumorspheroidimages,andmeasurecalceinAMandPIfluorescentintensitiesoftreatedU87MGMCTS

CeligoSetupPlateType NexcelomU-bottomUltra-lowAttachment384-wellPlate(Cat#ULA-384U)ScanChannels Green,Red,andBrightFieldResolution 1µm/pixelScanArea WholewellAnalysisMethod Tumorsphere1+2+MaskScanFrequency EndpointScanTime ~8min



ImageandanalyzethecytotoxicityeffectsofapanelofdrugcompoundsonU87MGMCTSonday13.


21. Seeded500U87MGcells/wellinULA384-wellplates22. Onday4,addeddifferentdiluteddrugcompoundsat2xandavehiclecontrolinmedia23. Onday13,preparedthecalceinAMandPIforstainingtheMCTS24. Incubatedtheplateat37°Cand5%CO2for60min25. ImagedandanalyzedonCeligo26. Comparedthespheroidviabilityforeachdrugcompoundateachtimepointtocharacterizethetested

compounds

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 18 20 21 22 23 24

A

B 10µM


CNTL

C 5µM

D 2.5µM

E 1µM

F 0.5µM

G 0.1µM

H 0.05µM

I 10µM


J 5µM

K 2.5µM

L 1µM

M 0.5µM

N 0.1µM

O 0.05µM

P


DataCollection

28. Afterincubatingthespheroidswithdrugsatdifferentconcentrations,thespheroidswerestainedwithcalceinAMandPI

29. Theplatewithstainedspheroidswasimagedonday1330. ThecapturedimageswerethenanalyzedintheCeligosoftwarefortheentire384-wellplate

DataAnalysis

• TheimageswereanalyzedbyusingTumorsphere1+2+MaskapplicationtoidentifytheMCTSinthewell

• ThefluorescentintensitiesofcalceinAMandPIweremeasuredforeachMCTStreatedwithdrugcompoundsorcontrol

Results

1.EndpointbrightfieldandfluorescentimagesandresultsofMCTSforcontrolandtreatedsample• Thebrightfieldimageswereusedtoidentifythespheroidsineachwell• ThecalceinAMandPIfluorescentintensitiesweremeasuredfromtheimages• CalceinAM/PIintensityratioswerecalculatedtodeterminetheviability• Compound3and4werehighlycytotoxictothetumorspheroids,whilecompound5and12reducedthe

sizeofthespheroidsbutdidnotinducecytotoxicity


• TheplotbelowshowsthecalceinAM/PIfluorescentintensityratios,whichindicatetheviabilityofeachMCTStreatedwiththedifferentdrugcompounds

• FewdrugcompoundshadmoderatetohighcytotoxiceffectsontheU87MGMCTS,whileotherdrugshadnoeffects

• Inadditiontoviabilitymeasurements,thespheroidsizecanalsoindicatesomecytotoxicityorgrowthinhibitioneffectsfromthedrugcompounds


analyzedthedatausingtheCeligoinstrument• Theentire384-wellplatewasimagedin~8min.Theshortscantimesignificantlyincreasedthe

throughputduringtheexperimentwhichhadmultipleplates• Afterthedrugtreatments,thespheroids’calceinAMandPIfluorescentintensitiesweremeasuredand

automaticallyreportedbytheCeligosoftware• NoadditionalsoftwarewasrequiredforimageanalysisofMCTSfluorescentintensities• Theabilitytomeasurefluorescencefromviabilitystains,incombinationwithspheroidsize,allowed

researcherstounderstandtheeffectsofdrugsonaspheroidlevel,whereenzymaticreadoutusingaplatereadermightnotprovide

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

CNTL 1 2 3 4 5 6 7 8 9 10 11 12 13 14

AVECA

MIN

T/AV

EPIIN

T

Compounds

CeligoDemonstrationExperiment–3DMCTSKineticPIAssay1361001426 Rev A

AssayName:3DMCTSKineticPropidiumIodideAssayAssayID:Celigo_03_0008


Experiment:3DMulticellularTumorSpheroidKineticPropidiumIodideAssayPurpose Kineticallymonitormulticellulartumorspheroid(MCTS)growth&Propidium

Iodide(PI)deadcellsignalonU87MGGlioblastomaovermulitpledaysCurrentMethod(s) Microscopyasanendpointassay,thirdpartyanalysisTargetCellType U87MGExperimentPlan AllowU87MGspheroidstoformwithPI,thenimagefor10daystomeasurethe

PIdeadcellsignalintensityandgrowthofspheresHypothesis Usingbrightfieldandfluorescentimaging,Celigowillprovideaneasy&rapid

methodtomonitormulticellulartumorspheroidgrowthandPIdeadcellsignalintensitiesforU87MGMCTS.

CeligoSetupPlateType NexcelomU-bottomUltra-LowAttachment96-wellplate(Cat#ULA-96U)ScanChannels Brightfield(BF),RedResolution 1µm/pixelScanArea WholewellAnalysisMethod Tumorsphere1+MaskScanFrequency Every24hoursScanTime ~2minutes



MonitorthespheroidgrowthandintensityofPIinU87MGMCTSfor10days

Protocol:27. Seededdifferentcellnumbersfrom3,200to100U87MGcells/wellinaULAU-bottom96-wellplate28. Added2XPIstainingsolutionandvehiclecontrolinmediatotheappropriatewells29. Incubatedtheplateat37°Cand5%CO230. ImagedandanalyzedonCeligofor10days31. AnalysedthePIintensitiesandsizeofthespheresfor10days

DataCollection

• AfterformingMCTSwithPI,theplatewasimagedinBFandRedilluminationsandanalyzeddailyfor10days.Eachscantook~2minutes.

DataAnalysis

• TheimageswereanalyzedbyusingTumorsphere1+Maskapplication• ThePIfluorescentintensitiesandspherediametersweremeasuredfortheMCTS

PlatemapforPIandControladdition1 2 3 4 5 6 7 8 9 10 11 12

ABCDEFGH

PI1µg/mL Control


ImageResultsFigure1.BrightfieldandredfluorescentimagesofU87MGMCTSwithPI(red)over10days

GraphsResultsFigure2.PIdeadcellsignalintensitesandspherediametersover10days.

LinegraphshowingthetrendsofPIintensityfordifferentcellconcentrationfor10days(a).LinegraphshowingthatthereisnotoxiceffectofPIonMCTSdiameter(b).

(a) (b)

Conclusion• UsingCeligoinstrument,imagesofU87MGMCTSweresuccessfullycapturedandanalyzedforgrowth

andPIdeadcellsignalintensities• AddingPIatthebeginningoftheexperimentallowedforkineticmonitoringofcelldeathinthecoreof

theMCTS.• Asthespherediameterbegantoplateau,thesignalofPIincreased• Theentire96-wellplatewasimagedin2minutes.Theshortscantimesignificantlyincreasedthe

throughputduringanexperimentthathadmultipleplates

CeligoDemonstrationExperiment–TranswellMigrationusingDAP1401001346 Rev B

AssayName:TranswellmigrationusingDAPIAssayID:Celigo_04_0001


Experiment:TranswellmigrationusingDAPIPurpose Imageandcountthenumberofcellswhichhavemigratedthroughthe

membraneofatranswellinsertusingfluorescentdetectionofDAPI-stainedcells.CurrentMethod(s) ManualvisualizationusingstandardlightmicroscopeTargetCellType Threedifferentcancercelllines(undisclosed)ExperimentPlan ScanplateusingBrightfieldandBluechannelsHypothesis Wewillbeabletostainandcountthenumberofcellswhichhavemigrated

throughthemembraneofatranswellinsert.

CeligoSetupPlateType Transwellinsertsplacedina24-wellBDFalcon353047plateScanChannels BrightfieldandBluechannelsResolution 1µm/pixelScanArea PartialWell-25FOVAnalysisMethod Target1+2ScanFrequency EndPointScanTime 3minutes

AssayProtocolandPlateSetup1. Threedifferentcancercelllineswereseededintoatranswellinsertandgrownaccordingtothecustomer’s

standardprocedure,withorwithouttreatmenttostimulatemigration.2. 24hrsafterseeding,thetranswellinsertswereremovedandthecellswerefixedinthetranswellinsert

withformaldehydefor10minutes.3. Cellswerethenwashedwithwatertoremovetheformaldehyde.4. Usingasterilecottonswap,cellswhichhadnotmigratedthroughthemembranewerescrapedoffthe

topofthetranswellinsert.5. AstainingsolutionofDAPI,withapermeabilityreagent,TritonX-100,waspreparedin5mLPBS.

a. 1:1,000dilutionofDAPIstock(10mg/mL)forafinal10µg/mLi. Nexcelom,Cat#CS1-0127

b. AddTritonX-100forafinal1%solution6. 600µLofstainingsolutionwasaddedtoacleanwellofa24-wellBDFalconplate(Cat#353047).7. Placedtranswell insert intoawellwithstainingsolutionandallowedtostain forapproximately10-15

minutes.8. RemovedtranswellinsertandwashedwithPBStoremoveanystainorTritonX-100.9. Added600µLofPBStoacleanwellofa24-wellBDFalconplate.10. Placedwashedtranswellinsertintothewell.11. ImagedonCeligousingtheTarget1+2applicationwithBrightfieldandBluechannels.


Results1. BrightfieldandBlueimageofwellA1

Fullinsertimages/partialwellimages(25FOV)

Zoomed-inimages

Note:ThecirclularobjectsseenintheBrightfieldimagesaretheporesinthetranswellmembrane.Theredarrowpointstooneofthemanyporesthatcanbeseenintheimage.


OverlayofBrightfieldandBlueimages

2. NumericalcountsofDAPI-positivecells

Note:TheoverlayofthebrightfieldimagewiththeDAPIimageallowstheconfirmationthatthecircularobjectsseeninthebrightfieldimagearetheporesinthemembrane,andnotcells(seeredarrow).ThecellsarestainedwithDAPIandappearasthebluedotsintheimage(seewhitearrow).Somecellsarestillmigratingthroughthepores,thusthebluedotsoccasionallylineupwithsomeoftheporesintheimage(seeyellowarrow).


Conclusion• TheCeligosuccessfullyimagedandcountedthenumberofDAPI-positivestainedcellswhichhad

migratedthroughamembraneinatranswellinsert.• AcquisitionofhighresolutionDAPIandbrightfieldimagesofthetranswellmembranesurfaceina

24wellplateformattook~3minutes.

CeligoDemonstrationExperiment–2DCellmigrationassayusingOrisTMPlatypusPlate1451001421 Rev A

AssayName:2DCellMigrationAssayusing96-wellOrisTMPlatypusPlateAssayID:Celigo_04_0004


Experiment:

Purpose ValidatewoundhealingmigrationassaywithOrisTMPlatypusplateonCeligoCurrentMethod(s) PlateReaderabsorbancereadingTargetCellType HT1080ExperimentPlan PlatecellsinOrisTMPlatypusPlate,removeplugs,adddrug,imageafter32hours.Hypothesis Inhibitcellmigration(woundhealing)withCytochalasinDdrugaddition.

CeligoSetupPlateType 96-wellPlatypusOris™CellMigrationAssayPlateCat#CMA1.101

Blackwall,clearbottomScanChannels BrightfieldchannelResolution 1µm/pixelScanArea WholewellAnalysisMethod WoundHealingScanFrequency OnceScanTime Lessthan10minutes

AssayProtocolandPlateSetupGoal

Measurethemigrationofdrug-treatedadherentHT1080cellsusing96-wellOris™Platypusplates

Protocol

Cellpreparation

33. Adherentcells(HT1080)wereplatedinOris™platewithplugandallowedtoadhereovernight.34. Theplugwasremovedanddrugwasaddedtowells.35. Theplatewasimagedat32hourspost-treatment.36. Cellcountsandconfluenceareawerereported.

PlatemapofCytochalasinD(µM)treatment

Min1 2 3 4 5 6 7 8 9 10 11 12

A 5 5 5 5 5 5 0 0 0 0 0 0

B 5 5 5 5 5 5 0 0 0 0 0 0

C 5 5 5 5 5 5 0 0 0 0 0 0

D 5 5 5 5 5 5 0 0 0 0 0 0

E 5 2.5 1.25 0.625 0.313 0.156 0.078 0.039 0.020 0.010 0.005 0

F 5 2.5 1.25 0.625 0.313 0.156 0.078 0.039 0.020 0.010 0.005 0

G 5 2.5 1.25 0.625 0.313 0.156 0.078 0.039 0.020 0.010 0.005 0

H 5 2.5 1.25 0.625 0.313 0.156 0.078 0.039 0.020 0.010 0.005 0

Max


DataCollection

31. Imagedwholewellandwholeplateat32hourspost-drugtreatmentwithWoundHealingApplicationinBrightfieldchannelinlessthan10minutes.

DataAnalysis

• AnalysisGraphicoverlaysforcells,woundandwellmaskshowedthatwoundareaandcellcountswereproperlydetected.Thewellmaskwasdecreasedtofocusanalysislocallyontheareaofthewoundcreatedbytheplug.

MergedWound

CellsWellMask


ResultsCeligo

• Wholeplateresultsshoweddatacoloredwith“heatmap”featuretoshowhigh,medium,andlowvaluesinImageview(top)andFillview(bottom)pseudo-colorfillinginareaofcellsdetectedinimage.


• WellLevelresultsshowedControl(left)andCytochalasinDtreatedwellsafter32hours(right)withfillview(green).

• DoseresponsecurveandrobustnessZfactorforCytochalsinDtreatment

Conclusion• Cellmigration(woundhealing)assaywithHT1080cellsplatedintheOris™Platypusplatewasimaged

andanalyzedonCeligoinlessthan10minutes.• TheCeligo-generateddatareportedcellcountsandwoundhealingareaforeachwelloveradrugcourse

treatment.TheCeligoimagecytometerproducedmorecomprehensiveimageandquantificationdatathanaplatereader.

• Doseresponsecurveandrobustnessfactor(Z’)calculatedtoshowrobustnessandcanbeperformedinordertovalidatethisassayforuseinyourlab.

Control CytochalasinD(5µM)

W o u n d H e a lin gH T 1 0 8 0 t re a te d w ith C y to c h a la s in D

-3 -2 -1 0 10

2 0

4 0

6 0

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d

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2 0

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6 0

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Documents

Celigo Demonstration Experiments - Nexcelom Bioscience · 2018-06-10 · Celigo Demonstration Experiment – NK cell-mediated cytotoxicity using calcein AM 9 1001321 Rev B Data Collection