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
CeligoDemonstrationExperiment–NKcell-mediatedcytotoxicityusingcalceinAM 71001321 Rev B
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
CeligoDemonstrationExperiment–NKcell-mediatedcytotoxicityusingcalceinAM 81001321 Rev B
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
CeligoDemonstrationExperiment–NKcell-mediatedcytotoxicityusingcalceinAM 91001321 Rev B
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
CeligoDemonstrationExperiment–NKcell-mediatedcytotoxicityusingcalceinAM111001321 Rev B
• 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
CeligoDemonstrationExperiment–NKcell-mediatedcytotoxicityusingcalceinAM121001321 Rev B
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
CeligoDemonstrationExperiment–NKcell-mediatedcytotoxicityusingcalceinAM131001321 Rev B
• 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
CeligoDemonstrationExperiment–NKcell-mediatedADCCusingcalceinAM151001324 Rev B
Experiment:NKcell-mediatedADCCusingcalceinAM
Purpose Measureantibody-dependentNK92cell-mediatedcytotoxicityusingcalceinAM-stainedMDA-MB-231
CurrentMethod(s) LDHReleaseAssayTargetCellType Target:MDA-MB-231;Effector:NK92cellsExperimentPlan ScanplateusingBrightfieldandGreenFluorescentchannelHypothesis BymeasuringthechangesinthenumberofcalceinAM-positivecellsovertime,
the%cytotoxicitycanbecalculatedusingtime0andcontrolfornormalization
CeligoSetupPlateType Greiner65509096-wellblackwallclearbottomScanChannels BrightfieldandGreenResolution 1µm/pixelScanArea WholewellAnalysisMethod Target1+2ScanFrequency Every2hours,upto6hoursScantime ~4min
CeligoDemonstrationExperiment–NKcell-mediatedADCCusingcalceinAM161001324 Rev B
AssayProtocolandPlateSetup
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
CeligoDemonstrationExperiment–NKcell-mediatedADCCusingcalceinAM171001324 Rev B
DataCollection
1. Afteraddingthecellsandantibodies,theplatewascentrifugedtosettlethecellstothebottom2. Immediatelyafter,theplatewasscannedinCeligousingTarget1(Green)+2(BF)fort=0h3. Repeatedthescanningfort=2,4,and6h
DataAnalysis
• TheimagesforeachtimepointwereanalyzedtocounttotalnumberofcalceinAM-positivecellsineachwell
• 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
CeligoDemonstrationExperiment–NKcell-mediatedADCCusingcalceinAM181001324 Rev B
Results1.Celigo-capturedcalceinAMfluorescentimagesatdifferenttimepoints
• Belowisanexampleofplateviewintheresultssectionshowingcellscountedontheplate
• BelowareexamplecalceinAMimagesforMDA-MB-231atdifferenttimepoints• ThenumberofcalceinAM-positivecellsdecreasedastimeincreased• ThepurpleoutlinesindicatethecountedcalceinAM-positivecells
CeligoDemonstrationExperiment–NKcell-mediatedADCCusingcalceinAM191001324 Rev B
• 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
CeligoDemonstrationExperiment–NKcell-mediatedADCCusingcalceinAM201001324 Rev B
• BelowareexamplecalceinAMandbrightfieldoverlayimagesat6hour,whichshowedtheformationofimmunecomplexclustersatdifferentantibodydosages
Conclusion• TheCeligowasabletomeasureADCCforNK92andMDA-MB-231bycountingthenumberofcalcein
AM-positivecellsovertime• ThenumberofcalceinAM-positivecellsdecreasedasantibodydosageandtimeincreased• Inaddition,theabilitytoviewbrightfieldimagesallowedobservationofformationofimmune
complexesindicatingcytotoxicity
CeligoDemonstrationExperiment–PBMC-mediatedcytotoxicityusingcalceinAM211001327 Rev B
AssayName:PBMC-mediatedcytotoxicyusingcalceinAMAssayID:Celigo_01_0011
CeligoDemonstrationExperiment–PBMC-mediatedcytotoxicityusingcalceinAM221001327 Rev B
Experiment:PBMC-mediatedcytotoxicityusingcalceinAM
Purpose MeasurePBMC-mediatedcytotoxicitybycountingtotalliveK562cellsovertimeinthepresenceandabsenceofIL2
CurrentMethod(s) ChromiumReleaseAssay,FlowCytometryTargetCellType Target:K562;Effector:PBMCsExperimentPlan ScanplateusingBrightfieldandGreenFluorescentchannelHypothesis BymeasuringthechangesinthenumberofcalceinAM-positivecellsovertime,
the%cytotoxicitycanbecalculatedusingtime0andcontrolfornormalization
CeligoSetupPlateType Greiner65509096-wellblackwallclearbottomScanChannels BrightfieldandGreenResolution 1µm/pixelScanArea WholewellAnalysisMethod Target1+2ScanFrequency Hourly,upto4hoursScanTime ~7min
CeligoDemonstrationExperiment–PBMC-mediatedcytotoxicityusingcalceinAM231001327 Rev B
AssayProtocolandPlateSetup
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
CeligoDemonstrationExperiment–PBMC-mediatedcytotoxicityusingcalceinAM241001327 Rev B
DataCollection
1. AfteraddingtheTargetandEffectorcellsandIL2,theplatewascentrifugedtosettlethecellstothebottom
2. 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• Normalizedtot=0
o %𝐶𝑦𝑡𝑜𝑡𝑜𝑥𝑖𝑐𝑖𝑡𝑦 𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑𝑡𝑜𝑡𝑖𝑚𝑒 = 1 − -./012345067389JK-./012345067389JL
• Normalizedtospontaneousreleaseo %𝐶𝑦𝑡𝑜𝑡𝑜𝑥𝑖𝑐𝑖𝑡𝑦 = %𝑐𝑦𝑡𝑜𝑡𝑜𝑥𝑖𝑐𝑖𝑡𝑦 𝑠𝑎𝑚𝑝𝑙𝑒 − %𝑐𝑦𝑡𝑜𝑡𝑜𝑥𝑖𝑐𝑖𝑡𝑦(𝑠𝑝𝑜𝑛𝑡)
• The%cytotoxicitywascalculatedforeverywellandaveragedtogenerateE:Tratio-dependentresponseandtime-coursemonitoring
CeligoDemonstrationExperiment–PBMC-mediatedcytotoxicityusingcalceinAM251001327 Rev B
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
CeligoDemonstrationExperiment–PBMC-mediatedcytotoxicityusingcalceinAM261001327 Rev B
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
CeligoDemonstrationExperiment–PBMC-mediatedcytotoxicityusingcalceinAM271001327 Rev B
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
CeligoDemonstrationExperiment–EndpointapoptosisusingCaspase3/7withHoechst 291001318 Rev B
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
CeligoDemonstrationExperiment–EndpointapoptosisusingCaspase3/7withHoechst 301001318 Rev B
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
CeligoDemonstrationExperiment–EndpointapoptosisusingCaspase3/7withHoechst 311001318 Rev B
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:
CeligoDemonstrationExperiment–EndpointapoptosisusingCaspase3/7withHoechst 321001318 Rev B
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
CeligoDemonstrationExperiment–EndpointviabilityusingDRAQ7andHoechst 341001360 Rev B
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
CeligoDemonstrationExperiment–EndpointviabilityusingDRAQ7andHoechst 351001360 Rev B
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
Hoechstaddedat48hrtimepoint
Hoechstaddedat72hrtimepoint
PositiveControl
DRAQ7(+)
CeligoDemonstrationExperiment–EndpointviabilityusingDRAQ7andHoechst 361001360 Rev B
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
CeligoDemonstrationExperiment–KineticViabilityusingDRAQ7 381001361 Rev B
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
CeligoDemonstrationExperiment–KineticViabilityusingDRAQ7 391001361 Rev B
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
CeligoDemonstrationExperiment–KineticViabilityusingDRAQ7 401001361 Rev B
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
MDA-MB-231Adherent K562Suspension
CeligoDemonstrationExperiment–HTsuspensioncellcountandviabilityusingAO/PI 411001369 Rev B
AssayName:HTsuspensioncellcountandviabilityusingAO/PIAssayID:Celigo_02_0007
CeligoDemonstrationExperiment–HTsuspensioncellcountandviabilityusingAO/PI 421001369 Rev B
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
CeligoDemonstrationExperiment–HTsuspensioncellcountandviabilityusingAO/PI 431001369 Rev B
AssayProtocolandPlateSetup
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
CeligoDemonstrationExperiment–HTsuspensioncellcountandviabilityusingAO/PI 441001369 Rev B
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
CeligoDemonstrationExperiment–HTsuspensioncellcountandviabilityusingAO/PI 451001369 Rev B
Results1.Celigo-capturedJurkatcellconcentrationseriesAOfluorescentimages
• BelowareexampleAOfluorescentimagesatdifferentconcentrations
CeligoDemonstrationExperiment–HTsuspensioncellcountandviabilityusingAO/PI 461001369 Rev B
• 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
CeligoDemonstrationExperiment–HTsuspensioncellcountandviabilityusingAO/PI 471001369 Rev B
2.Celigo-capturedJurkatcellviabilityseriesAO/PIfluorescentimages• BelowareexampleAO/PIfluorescentimagesatdifferentviabilities• ThenumberofAOpositivecellsdecreasedastheviabilitydecreased• ThenumberofPIpositivecellsincreasedastheviabilitydecreased
CeligoDemonstrationExperiment–HTsuspensioncellcountandviabilityusingAO/PI 481001369 Rev B
• 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
CeligoDemonstrationExperiment–EndpointviabilityusingPIandHoechst 501001355 Rev B
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
AssayProtocolandPlateSetup
Goal:DetectandquantifydeadcellsusingPIandHoechststainsinadherentMDA-MB-231andsuspensionK562celllines
Protocol
• SeededMDA-MB-231andallowedtoincubateovernight.K562Suspensioncellswereplatedonthedayoftheexperiment
• PreparedandseriallydilutedthedrugBenzethoniumtogenerateadoseresponse• Preparedthecontrolwithwaterinmedia• Addeddrugdoseresponseandcontroltothewells• Incubatedtheplatefor24,48and72hours• PreparedadyemixsolutionofPIandHoechstinPBS• Addeddyemixtothedrug-treatedwellsandincubatedtheplate• ImagedtheplateusingCeligoimagecytometer
CeligoDemonstrationExperiment–EndpointviabilityusingPIandHoechst 511001355 Rev B
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+
CeligoDemonstrationExperiment–EndpointviabilityusingPIandHoechst 521001355 Rev B
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
CeligoDemonstrationExperiment–KineticviabilityusingPI 541001358 Rev B
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
CeligoDemonstrationExperiment–KineticviabilityusingPI 551001358 Rev B
ResultsDrug-treatedMDA-MB-231andK562cellsshowedanincreaseinPIpositivecells
• PI-positivecellsweredeterminedbystainingthecellsfor24,48and72hours
TypicalimagesandfluorescentobjectidentificationlookedasshownbelowforPIstainedcells“GraphicOverlay”segmentation
ResultsfortheK562andMDA-MB-31countsofdeadcellsafter24hoursofBenzethoniumdrugtreatment
MDA-MB-231Adherent K562Suspension
BF+RedImage RedImage BFImageImages+GraphicOverlay
K562
-Su
spen
sion
MDA
-MB-23
1-A
dheren
t
CeligoDemonstrationExperiment–KineticviabilityusingPI 561001358 Rev B
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
CeligoDemonstrationExperiment–KineticApoptosisusingCaspase3/7 581001388 Rev A
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
CeligoDemonstrationExperiment–KineticApoptosisusingCaspase3/7 591001388 Rev A
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
CeligoDemonstrationExperiment–KineticApoptosisusingCaspase3/7 601001388 Rev A
Graphs
6. InMicrosoftExcel,createaveragesandstandarddeviationsofthecontrolanddrug-treatedwells.
7. Generatea“Bargraph”comparing3µMStaurosporinetothecontrolover8hourtimecourse.Inthisexample,theaverageof12datapointswereplotted.
CeligoDemonstrationExperiment–KineticApoptosisusingCaspase3/7 611001388 Rev A
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
CeligoDemonstrationExperiment–HPCproliferationmeasurementusingKi-67 631001381 Rev A
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
CeligoDemonstrationExperiment–HPCproliferationmeasurementusingKi-67 641001381 Rev A
AssayProtocolandPlateSetup
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
CeligoDemonstrationExperiment–HPCproliferationmeasurementusingKi-67 651001381 Rev A
Results1.Celigo-capturedbrightfield,DyLight288andDAPIfluorescentimages
• Examplesofbrightfield,Ki-67-DyLight488andDAPI-stainedfluorescentimages
• CeligowasabletocountDAPI-positivecells,todeterminethetotalpopulation,usethegatingfunctiontoidentifyKi-67-positivecellsandcalculatethepercentofKi-67-positivecellsforthewholeplate
BrightField Ki-67-DyLight488 DAPI
CeligoDemonstrationExperiment–HPCproliferationmeasurementusingKi-67 661001381 Rev A
• 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
CeligoDemonstrationExperiment–Antibody-DependentReceptorInternalizationAssay 681001390 Rev A
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
CeligoDemonstrationExperiment–Antibody-DependentReceptorInternalizationAssay 691001390 Rev A
AssayProtocolandPlateSetup
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
CeligoDemonstrationExperiment–Antibody-DependentReceptorInternalizationAssay 701001390 Rev A
Results1.ReceptorInternalizationimagesusingCeligoImagingCytometer
• Celigowasusedtocapturebrightfieldandfluorescentwhole-wellimageson96-wellplates• Itrequired~9min/plateforimageacquisitionandfluorescentdataanalysis• Wholeplateoverviewcanbeviewedtoquicklyassessthereceptorinternalizationresults(Seefigure
below)o Inthisexample,weshowedaplateviewofHT-293at5hourontheCeligosoftwaretoprovidea
quickat-a-glanceresultofthereceptorinternalizationo Thevaluesshownineachwellarethetotalorintegratedfluorescentintensities
CeligoDemonstrationExperiment–Antibody-DependentReceptorInternalizationAssay 711001390 Rev A
• Eachwellcanbefurtherzoomed-intoviewimagesofindividualwellsandcellpopulationsinthewells,whichallowsvisualconfirmation(Seefigurebelow)
o Inthisexample,weshowedHT-293,zoomed-inviewat5houranddifferentAntibodytreatmentconcentrations
o Itisclearthattheredfluorescenceincreasedastheconcentrationofpositiveantibodyincreased
CeligoDemonstrationExperiment–Antibody-DependentReceptorInternalizationAssay 721001390 Rev A
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
CeligoDemonstrationExperiment–Antibody-DependentReceptorInternalizationAssay 731001390 Rev A
Conclusion• Theresultsshowedacleardifferencebetweennegativeantibodyandpositiveantibodyin
internalization• Adoseresponsewasobservedforpositiveantibodybymeasuringaveragetotalfluorescentintensity• ByusingthebackgroundcorrectionfunctiononCeligo,thebackgroundcanberemovedtoincreasethe
fluorescentintensities
CeligoDemonstrationExperiment–Countandmeasurecellinfectivityofmicrocarriers 741001399 Rev A
AssayName:CountandmeasurecellinfectivityofmicrocarriersAssayID:Celigo_02_0016
CeligoDemonstrationExperiment–Countandmeasurecellinfectivityofmicrocarriers 751001399 Rev A
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
CeligoDemonstrationExperiment–Countandmeasurecellinfectivityofmicrocarriers 761001399 Rev A
AssayProtocolandPlateSetup
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
CeligoDemonstrationExperiment–Countandmeasurecellinfectivityofmicrocarriers 771001399 Rev A
Results1.Celigo-capturedbrightfieldandfluorescentimagesofDAPIandAlexaFluor488
• TheCeligowasabletomeasure%infectivitybycountingtotalnumberofDAPIandAlexaFluor488positivecells
• BydividingAlexaFluorbyDAPI,the%infectivitywascalculated
CeligoDemonstrationExperiment–Countandmeasurecellinfectivityofmicrocarriers 781001399 Rev A
• Thewholewellimageshowedallthemicrocarriersinthewellandwerecounted
• Whenzoomedin,thecoverageofcellscanbeclearlyobservedintwodifferentsamples,B4andB12
CeligoDemonstrationExperiment–Countandmeasurecellinfectivityofmicrocarriers 791001399 Rev A
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
CeligoDemonstrationExperiment–Countandmeasurecellinfectivityofmicrocarriers 801001399 Rev A
3.Celigo-capturedbrightfieldandfluorescentimagesofcellsandmicrocarriersforHoechstandPI• TheCeligowasusedtocapturebrightfieldimagesofthemicrocarriersforcounting• TheCeligowasalsousedtocaptureHoechstandPIstainedcellsgrowingonthemicrocarriersinorderto
measurethetotalcellcountsperwell,andpermicrocarrier• IndividualHoechstandPI-positivecellswerecounteddirectlyinthe96-wellplate,aswellasthe
microcarriers,shownintheimagesbelow.
CeligoDemonstrationExperiment–Countandmeasurecellinfectivityofmicrocarriers 811001399 Rev A
CeligoDemonstrationExperiment–Countandmeasurecellinfectivityofmicrocarriers 821001399 Rev A
• 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
CeligoDemonstrationExperiment–GFPTransfectionEfficiencyMeasurement 841001403 Rev A
Experiment:GFPTransfectionEfficiencyMeasurement
Purpose Toanalyzetransfectionefficiencyovera4dayperiodwhentreatedwithvariousamountsofatransfectioncompound.
CurrentMethod(s) ManualobservationusingfluorescentmicroscopeTargetCellType 293HcellsExperimentPlan Platetransfectedcellsandmeasuretransfectionefficiencyfor4daysHypothesis HigherviraldosagetreatmentswillleadtohigherGFPnumbersandfluorescent
intensities
CeligoSetupPlateType Greiner65509096-wellblackwallclearbottomScanChannels Brightfield+GreenResolution 1µm/pixelScanArea WholewellAnalysisMethod ConfluenceRatio:Confluence1+2ScanFrequency DailyScanTime ~8min
CeligoDemonstrationExperiment–GFPTransfectionEfficiencyMeasurement 851001403 Rev A
AssayProtocolandPlateSetup
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
CeligoDemonstrationExperiment–GFPTransfectionEfficiencyMeasurement 861001403 Rev A
DataCollection
14. Aftercentrifugingtheplate,theplatewasscannedusingtheCeligo15. Thescanningparametersfor2channelsweresetup,whereConfluencechannel1+2areGFPand
brightfield,respectively
DataAnalysis
• Theimageswereanalyzedtomeasuretotalareaofcellcoverageinbrightfieldandinfluorescence• TheautomaticallycalculatedconfluenceratioindicatesGFPtransfectionefficiencyasapercentageof
thetotalcellarea
Results1.GFPexpressionfluorescentimages
• TheGFPfluorescentimagesshoweddifferencesbetweenlowandhighcompoundtreatment• TheGFPexpressionplateviewisshownbelow
CeligoDemonstrationExperiment–GFPTransfectionEfficiencyMeasurement 871001403 Rev A
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
CeligoDemonstrationExperiment–GFPTransfectionEfficiencyMeasurement 881001403 Rev A
3.Brightfieldconfluencelevel• Thebrightfieldimagesshowedthatathighcompoundtreatment,thereisreductionincellconfluency,
incomparisontothelowcompoundtreatment
CeligoDemonstrationExperiment–GFPTransfectionEfficiencyMeasurement 891001403 Rev A
• 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
CeligoDemonstrationExperiment–GFPTransfectionEfficiencyMeasurement 901001403 Rev A
Conclusion• TheGFPtransfectionefficiencieswerehighlydependentonthecompoundtreatment• Atlowerseedingdensity,thecellsshowedhigherdamageduetohighcompoundtreatment
CeligoDemonstrationExperiment–Antibody-DependantDrugUptakeAssay 911001404 Rev A
AssayName:Antibody-DependentDrugUptakeAssayAssayID:Celigo_02_0019
CeligoDemonstrationExperiment–Antibody-DependantDrugUptakeAssay 921001404 Rev A
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
CeligoDemonstrationExperiment–Antibody-DependantDrugUptakeAssay 931001404 Rev A
AssayProtocolandPlateSetup
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
CeligoDemonstrationExperiment–Antibody-DependantDrugUptakeAssay 941001404 Rev A
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
CeligoDemonstrationExperiment–Antibody-DependantDrugUptakeAssay 951001404 Rev A
Results1.Celigogatingfunctionforfluorescentintensityanalysis
• Cellimageswereanalyzedbycountingthetotalnumberofcells(Hoescht)andcellsweregatedbasedonthemeanfluorescentintensitytodeterminepercentofdruguptake
Control,untreated(AF488+Hoescht)
Drug2,Ab0.5(AF488+Hoescht)
Drug2,Ab20(AF488+Hoescht)
CeligoDemonstrationExperiment–Antibody-DependantDrugUptakeAssay 961001404 Rev A
2.Celigo-capturedAlexaFluor488fluorescentimagesforsuspensioncells
• Brightfieldandfluorescentimagesatdifferentdrugandantibodyconcentrationsforsuspensioncells• RepresentativeimagesofcellsincubatedwithDrug2and1atahigh(20)andlow(0.5)antibody
concentration
CeligoDemonstrationExperiment–Antibody-DependantDrugUptakeAssay 971001404 Rev A
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
CeligoDemonstrationExperiment–Antibody-DependantDrugUptakeAssay 981001404 Rev A
4.Celigo-capturedAlexaFluor488fluorescentimagesforadherentcells
• BrightfieldandfluorescentimagesatatdifferentDrugandAntibodyconcentrationsforadherentcells• RepresentativeimagesofcellsincubatedwithDrug2,1,and0.5atahigh(20)andlow(0.5)antibody
concentration
CeligoDemonstrationExperiment–Antibody-DependantDrugUptakeAssay 991001404 Rev A
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
CeligoDemonstrationExperiment–Label-freeTumorSpheroidGrowthInhibition1021001383 Rev A
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
CeligoDemonstrationExperiment–Label-freeTumorSpheroidGrowthInhibition1031001383 Rev A
AssayProtocolandPlateSetup
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
CeligoDemonstrationExperiment–Label-freeTumorSpheroidGrowthInhibition1041001383 Rev A
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
CeligoDemonstrationExperiment–Label-freeTumorSpheroidGrowthInhibition1051001383 Rev A
Day4posttreatmentwithDrugXatHypoxiaNCI-H640at2,500andMiaPacaat1,250cells/well
• Theentireplatewasscannedin3minutesandbrightfieldthumbnailsareshownforeachwell.• AtthebottomofthewellpictureistheCeligomeasureddiameterin(microns)foreachidentified
spheroid.
Celigocapturedrepresentativebrightfieldimagesofnon-drugtreatedNCI-H460andMiaPacamulticellulartumorspheroidsatnormoxicconditiononday0
MiaPaca NCI-H460
NCI-H4602,500Cells/w
ellMiaPaca
1,250Cells/well
CeligoDemonstrationExperiment–Label-freeTumorSpheroidGrowthInhibition1061001383 Rev A
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
CeligoDemonstrationExperiment–3DMCTSinvasionscreeningassay1081001391 Rev A
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
CeligoDemonstrationExperiment–3DMCTSinvasionscreeningassay1091001391 Rev A
AssayProtocolandPlateSetup
Goal:
ImageandanalyzetheinhibitoryeffectsofapanelofdrugcompoundsonU87MGMCTSinvasionintoBasementMembraneExtractovertime.
ProtocolCellPreparation
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
CeligoDemonstrationExperiment–3DMCTSinvasionscreeningassay1101001391 Rev A
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
CeligoDemonstrationExperiment–3DMCTSinvasionscreeningassay1111001391 Rev A
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
CeligoDemonstrationExperiment–3DMCTSinvasionscreeningassay1121001391 Rev A
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
400000
600000
800000
1000000
CNTL 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Invasio
nArea(µ
m2)
Compounds
AverageInvasionAreaat5µMofDrug
CeligoDemonstrationExperiment–3DMCTSinvasionscreeningassay1131001391 Rev A
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
CeligoDemonstrationExperiment–3DMCTSgrowthinhibitionscreeningassay1151001393 Rev A
Experiment:3Dmulticellulartumorspheroid(MCTS)growthinhibitionscreeningassay
Purpose MonitortheeffectsofapanelofdrugsonthegrowthinhibitionofU87MGGlioblastomaMCTS
CurrentMethod(s) MicroscopyTargetCellType U87MGExperimentPlan AllowU87MGspheroidstoformandtreatwithapanelofdrugcompounds,then
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
CeligoDemonstrationExperiment–3DMCTSgrowthinhibitionscreeningassay1161001393 Rev A
AssayProtocolandPlateSetupGoal:
ImageandanalyzethegrowthinhibitioneffectofapanelofdrugcompoundsonU87MGMCTSovertime.
ProtocolCellPreparation
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
Cmpd8 Cmpd9 Cmpd10 Cmpd11 Cmpd12 Cmpd13 Cmpd14
J 5µM
K 2.5µM
L 1µM
M 0.5µM
N 0.1µM
O 0.05µM
P
CeligoDemonstrationExperiment–3DMCTSgrowthinhibitionscreeningassay1171001393 Rev A
DataCollection
22. Afteraddingthedrugatdifferentconcentrationsonday4,theplateswereimagedanddatacollectedfortheentire384-wellplate
23. Theplateswereagainimagedon0,69,114,165,210hourspost-treatmentinordertoperformtimecoursemonitoringoftumorspheroiddiameter
DataAnalysis
• TheimageswereanalyzedbyusingtheTumorsphere1applicationtoidentifytheMCTSinthewell• ThediameterofeachMCTStreatedwithdifferentdrugcompoundswasmeasured
Results
1.Time-coursebrightfieldimagesandresultsofMCTSforcontrolandtreatedsample• ThebrightfieldimagesshowedthecontrolMCTSincreasedindiameterovertimewhilethetreated
samplewithcompound8showedgrowthinhibition
CeligoDemonstrationExperiment–3DMCTSgrowthinhibitionscreeningassay1181001393 Rev A
• Thetime-courseresultsshowedthatsomedrugsinhibitedthegrowthinthebeginningofthetreatment,andsomedrugsinhibitedseveraldaysafterthetreatment
• Somedrugcompoundsdidnotinduceinhibition,similartothecontrol
2.Dose-responsebrightfieldimagesandresultsofMCTSforcontrolandtreatedsample• Thebrightfieldimagesshowedthedoseresponseofcompound8growthinhibitionofMCTS• Astheconcentrationincreased,thespheroidsizedecreased
200
250
300
350
400
450
500
550
600
650
0 50 100 150 200 250
Sphe
roidDiameter(µ
m)
Time(hour)
Time-CourseMonitoringofSpheroidDiameter Control1234567891011121314
CeligoDemonstrationExperiment–3DMCTSgrowthinhibitionscreeningassay1191001393 Rev A
• 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
CeligoDemonstrationExperiment–3DMCTSgrowthinhibitionscreeningassay1201001393 Rev A
Conclusion• Usingthe384-wellU-bottomULAplates,wesuccessfullycapturedimagesofU87MGMCTSand
analyzedthedatausingtheCeligoimagingcytometer• Theentire384-wellplatewasimagedin~4min.Theshortscantimesignificantlyincreasesthe
throughputduringanexperimentthathasmultipleplates• Afterthedrugtreatments,spheroiddiametersweremeasuredandautomaticallyreportedbytheCeligo
software• NoadditionalsoftwarewasrequiredforimageanalysisofMCTSdiameters
CeligoDemonstrationExperiment–3DMCTSendpointapoptosisscreening1211001397 Rev A
AssayName:3Dmulticellular tumor spheroidendpoint apoptosisscreeningAssayID:Celigo_03_0005
CeligoDemonstrationExperiment–3DMCTSendpointapoptosisscreening1221001397 Rev A
Experiment:3Dmulticellulartumorspheroid(MCTS)endpointapoptosisscreeningassay
Purpose MonitortheeffectsofapanelofdrugsontheapoptosisofU87MGGlioblastomaMCTSusingCaspase3/7andHoechstfluorescentstaining
CurrentMethod(s) MicroscopyTargetCellType U87MGExperimentPlan AllowU87MGspheroidstoformandtreatwithapanelofdrugcompounds,then
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
CeligoDemonstrationExperiment–3DMCTSendpointapoptosisscreening1231001397 Rev A
AssayProtocolandPlateSetupGoal:
ImageandanalyzetheapoptoticeffectsofapanelofdrugcompoundsonU87MGMCTSonday13.
ProtocolCellPreparation
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
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
Cmpd8 Cmpd9 Cmpd10 Cmpd11 Cmpd12 Cmpd13 Cmpd14
J 5µM
K 2.5µM
L 1µM
M 0.5µM
N 0.1µM
O 0.05µM
P
CeligoDemonstrationExperiment–3DMCTSendpointapoptosisscreening1241001397 Rev A
DataCollection
24. Afterincubatingthespheroidswithdrugsatdifferentconcentrations,thespheroidswerestainedwithCaspase3/7andHoechst
25. Theplatewithstainedspheroidswasimagedonday1326. ThecapturedimageswerethenanalyzedintheCeligosoftwarefortheentire384-wellplate
DataAnalysis
• TheimageswereanalyzedbyusingTumorsphere1+2+MaskapplicationtoidentifytheMCTSinthewell
• ThefluorescentintensitiesofCaspase3/7weremeasuredforeachdrug-treatedMCTSorcontrol
Results
1.EndpointbrightfieldandfluorescentimagesandresultsofMCTSforcontrolandtreatedsample• Thebrightfieldimageswereusedtoidentifythespheroidsineachwell• TheCaspase3/7fluorescentintensitiesweremeasuredfromtheimages
CeligoDemonstrationExperiment–3DMCTSendpointapoptosisscreening1251001397 Rev A
• TheplotbelowisshowingtheCaspase3/7fluorescentintensities,whichindicatedtheapoptosisofeachMCTStreatedwiththedifferentdrugcompounds
• Only2drugcompoundsinducednoticeableapoptosisontheU87MGMCTS,whileotherdrugshadnoeffects
Conclusion• Usingthe384-wellU-bottomULAplates,wesuccessfullycapturedimagesofU87MGMCTSand
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
CeligoDemonstrationExperiment–Countingofpatient-derivediDCorganoids1271001400 Rev A
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
CeligoDemonstrationExperiment–Countingofpatient-derivediDCorganoids1281001400 Rev A
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
CeligoDemonstrationExperiment–Countingofpatient-derivediDCorganoids1291001400 Rev A
Results1.Celigo-capturedbrightfieldimagesoforganoids
• Celigocapturedbrightfieldimagesin12-wellplatescontainingorganoidsinmatrigel• Belowisanexamplewholeplateimageofthecaptureddata
• Celigocapturedhighresolutionimagesinbrightfieldthatcanbezoomed-inonfromwholewell(below)
CeligoDemonstrationExperiment–Countingofpatient-derivediDCorganoids1301001400 Rev A
• 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
CeligoDemonstrationExperiment–3DMCTSendpointviabilityscreeningassay1321001406 Rev A
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
CeligoDemonstrationExperiment–3DMCTSendpointviabilityscreeningassay1331001406 Rev A
AssayProtocolandPlateSetupGoal:
ImageandanalyzethecytotoxicityeffectsofapanelofdrugcompoundsonU87MGMCTSonday13.
ProtocolCellPreparation
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
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
Cmpd8 Cmpd9 Cmpd10 Cmpd11 Cmpd12 Cmpd13 Cmpd14
J 5µM
K 2.5µM
L 1µM
M 0.5µM
N 0.1µM
O 0.05µM
P
CeligoDemonstrationExperiment–3DMCTSendpointviabilityscreeningassay1341001406 Rev A
DataCollection
28. Afterincubatingthespheroidswithdrugsatdifferentconcentrations,thespheroidswerestainedwithcalceinAMandPI
29. Theplatewithstainedspheroidswasimagedonday1330. ThecapturedimageswerethenanalyzedintheCeligosoftwarefortheentire384-wellplate
DataAnalysis
• TheimageswereanalyzedbyusingTumorsphere1+2+MaskapplicationtoidentifytheMCTSinthewell
• ThefluorescentintensitiesofcalceinAMandPIweremeasuredforeachMCTStreatedwithdrugcompoundsorcontrol
Results
1.EndpointbrightfieldandfluorescentimagesandresultsofMCTSforcontrolandtreatedsample• Thebrightfieldimageswereusedtoidentifythespheroidsineachwell• ThecalceinAMandPIfluorescentintensitiesweremeasuredfromtheimages• CalceinAM/PIintensityratioswerecalculatedtodeterminetheviability• Compound3and4werehighlycytotoxictothetumorspheroids,whilecompound5and12reducedthe
sizeofthespheroidsbutdidnotinducecytotoxicity
CeligoDemonstrationExperiment–3DMCTSendpointviabilityscreeningassay1351001406 Rev A
• TheplotbelowshowsthecalceinAM/PIfluorescentintensityratios,whichindicatetheviabilityofeachMCTStreatedwiththedifferentdrugcompounds
• FewdrugcompoundshadmoderatetohighcytotoxiceffectsontheU87MGMCTS,whileotherdrugshadnoeffects
• Inadditiontoviabilitymeasurements,thespheroidsizecanalsoindicatesomecytotoxicityorgrowthinhibitioneffectsfromthedrugcompounds
Conclusion• Usingthe384-wellU-bottomULAplates,wesuccessfullycapturedimagesofU87MGMCTSand
analyzedthedatausingtheCeligoinstrument• Theentire384-wellplatewasimagedin~8min.Theshortscantimesignificantlyincreasedthe
throughputduringtheexperimentwhichhadmultipleplates• Afterthedrugtreatments,thespheroids’calceinAMandPIfluorescentintensitiesweremeasuredand
automaticallyreportedbytheCeligosoftware• NoadditionalsoftwarewasrequiredforimageanalysisofMCTSfluorescentintensities• Theabilitytomeasurefluorescencefromviabilitystains,incombinationwithspheroidsize,allowed
researcherstounderstandtheeffectsofdrugsonaspheroidlevel,whereenzymaticreadoutusingaplatereadermightnotprovide
0.00
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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
CeligoDemonstrationExperiment–3DMCTSKineticPIAssay1371001426 Rev A
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
CeligoDemonstrationExperiment–3DMCTSKineticPIAssay1381001426 Rev A
AssayProtocolandPlateSetupGoal:
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
CeligoDemonstrationExperiment–3DMCTSKineticPIAssay1391001426 Rev A
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
CeligoDemonstrationExperiment–TranswellMigrationusingDAP1411001346 Rev B
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.
CeligoDemonstrationExperiment–TranswellMigrationusingDAP1421001346 Rev B
Results1. BrightfieldandBlueimageofwellA1
Fullinsertimages/partialwellimages(25FOV)
Zoomed-inimages
Note:ThecirclularobjectsseenintheBrightfieldimagesaretheporesinthetranswellmembrane.Theredarrowpointstooneofthemanyporesthatcanbeseenintheimage.
CeligoDemonstrationExperiment–TranswellMigrationusingDAP1431001346 Rev B
OverlayofBrightfieldandBlueimages
2. NumericalcountsofDAPI-positivecells
Note:TheoverlayofthebrightfieldimagewiththeDAPIimageallowstheconfirmationthatthecircularobjectsseeninthebrightfieldimagearetheporesinthemembrane,andnotcells(seeredarrow).ThecellsarestainedwithDAPIandappearasthebluedotsintheimage(seewhitearrow).Somecellsarestillmigratingthroughthepores,thusthebluedotsoccasionallylineupwithsomeoftheporesintheimage(seeyellowarrow).
CeligoDemonstrationExperiment–TranswellMigrationusingDAP1441001346 Rev B
Conclusion• TheCeligosuccessfullyimagedandcountedthenumberofDAPI-positivestainedcellswhichhad
migratedthroughamembraneinatranswellinsert.• AcquisitionofhighresolutionDAPIandbrightfieldimagesofthetranswellmembranesurfaceina
24wellplateformattook~3minutes.
CeligoDemonstrationExperiment–2DCellmigrationassayusingOrisTMPlatypusPlate1451001421 Rev A
AssayName:2DCellMigrationAssayusing96-wellOrisTMPlatypusPlateAssayID:Celigo_04_0004
CeligoDemonstrationExperiment–2DCellmigrationassayusingOrisTMPlatypusPlate1461001421 Rev A
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
CeligoDemonstrationExperiment–2DCellmigrationassayusingOrisTMPlatypusPlate1471001421 Rev A
DataCollection
31. Imagedwholewellandwholeplateat32hourspost-drugtreatmentwithWoundHealingApplicationinBrightfieldchannelinlessthan10minutes.
DataAnalysis
• AnalysisGraphicoverlaysforcells,woundandwellmaskshowedthatwoundareaandcellcountswereproperlydetected.Thewellmaskwasdecreasedtofocusanalysislocallyontheareaofthewoundcreatedbytheplug.
MergedWound
CellsWellMask
CeligoDemonstrationExperiment–2DCellmigrationassayusingOrisTMPlatypusPlate1481001421 Rev A
ResultsCeligo
• Wholeplateresultsshoweddatacoloredwith“heatmap”featuretoshowhigh,medium,andlowvaluesinImageview(top)andFillview(bottom)pseudo-colorfillinginareaofcellsdetectedinimage.
CeligoDemonstrationExperiment–2DCellmigrationassayusingOrisTMPlatypusPlate1491001421 Rev A
• 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
L o g [u M ]
Pe
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Wo
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He
ale
d
C o n tr o l T r e a te d0
2 0
4 0
6 0
R o b u s tn e s s
Pe
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Wo
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He
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Z ' = 0 .5 7
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