G e m i n i B i o A p p l i c a t i o n N o t e 3 / 2 0 2 1

Rapid Apoptosis Monitoring Using Gemini’s Bio’s Moxi GO II and Apoptosis Kit

Introduction/BackgroundCellular apoptosis is a sophisticatedmechanismemployedbycellstocarefullycontrol death in response to cell injury.Commonly referred to as “programmedcelldeath,”apoptosisprogressesthroughasystematicsignalingcascadethatresultsin characteristic, directed morphologicalandbiochemicaloutputs in thecell. Theoveralloutcomeisahighlyregulatedcelldeath process that minimizes trauma tothecell’sextracellularenvironment.Atasystemic level, the critical role ofapoptosisiseasilyhighlightedthroughitsimplication in hearing loss1,cardiovascular failure2, cancerprogression3, neurodegenerativedisorders4, and numerous otherpathologies5. Correspondingly, scientificresearchhasintentlyfocusedonrevealingthe underlying pathways of apoptosis,defining its external triggers, identifyingtherapeutic interventions, and furtherexploringtheroleofapoptosisinnumerouspathologies.Furthermore,themonitoringofapoptosishasalsobeenestablishedasakeyindicatorinidentifyingthe“biocompatibility”ofpharmaceuticsandotherenvironmentalconditionsforcellsystems. Concurrentwiththeincreasedinterestinapoptosismonitoring,numerousassayshavebeencreatedto quantify the expression of associated molecular targets such as phosphatidylserine (PS)externalizationintheplasmamembraneandcaspaseenzymeactivationaswellasotherapoptoticcharacteristics including mitochondrial potential changes, chromatin condensation, and DNAfragmentation. However, these assays often require advanced technical expertise, costlyinstrumentation, and time-consuminganalysisusing techniques suchaswesternblot,ELISA, andflowcytometry.Here,wepresenttheGeminiBioMoxiGOIIsystemasasimple,rapid,andeffectiveflowcytometricapproachtothestudyofapoptosisusingAnnexinV.GeminiBio’snewest “NextGenerationFlowCytometer”, theMoxiGO II (Figure1), is ideally anduniquelysuitedtofulfillingresearchersneedsforapoptosismonitoring.ThesystemcombinestheCoulterPrinciple,therecognizedgoldstandardforprecisecellsizingandcounts,withsimultaneousfluorescentmeasurements using a 488nm laser, coupled with two PMT detection channels, onefilteredtodetectat525/45nmandtheotheratwith561nm/LP.Thisfluorescenceconfigurationisideal formany of themost common fluorophores including phycoerythrin (PE, immunolabeling,Annexin V), propidium iodide (PI for viability), Calcein-AM (Cell Health), FITC (immunolabeling,Annexin V), and GFP (transfection efficiency). The Moxi GO II utilizes a disposable flow-cellarchitecture, does not require warm-up, runs test in under 10 seconds, and does not requirecleaning/shutdownprocedures.Itslowcostandverysmallfootprintmakeiteasyforresearchers’toacquireone for theirown lab,placing iton thebench-toporeven thecell culturehood. Thisenablesresearcherstoeasilyacquiretemporalflowdataovershortorlongperiods.Theresultisanaffordableflowcytometerthatdelivers“AssaysonDemand,”includingapoptosismonitoringwithAnnexinV.

Figure1–Orflo’sMoxiGOII–NextGenerationFlowCytometer.TheMoxiGOIIisa configurable flow cytometer with a 488nm laser and up to two fluorescencerecording channels (2 PMT’s with emission filters at 525/45nm filter and561nm/LPOR646nm/LP(user-swappable),respectively).

G e m i n i B i o A p p l i c a t i o n N o t e 3 / 2 0 2 1

Rapid Apoptosis Monitoring Using Gemini’s Bio’s Moxi GO II and Apoptosis Kit

ExampleData–ResultsandDiscussionOneofthemostwell-characterized,early-stagemarkersinthecellularapoptosisprogressionisthetranslocationofthemembranephospholipid,phosphatidylserine(PS),fromtheinnertotheouterleafletoftheplasmamembrane.ThedetectionofPStranslocationisachievedthroughtheuseofAnnexinV,acellularproteinwithahighaffinityforPS.BecauseAnnexinViscell-impermeant,onlycellswithapoptosis-relatedexposureofPS,orcellswithcompromisedmembranes(necroticorlate-stageapoptoticcells),arelabeledwiththisprobe.Consequently,byconjugatingAnnexinVtoasuitablefluorophore(e.g.FITCfortheMoxiGOII),cellscanbelabeledforPSinordertodistinguishAnnexinV+(apoptotic)fromAnnexinV-(healthy)populations.Figure2aprovidesarepresentative,user-generatedscreenshotforaFITC-AnnexinVlabeledJurkatsamplethatwasprocessedontheMoxiGOII.ThelargefluorescenceshiftsfortheAnnexinV+vs.AnnexinV-andPI+vs.PI-cellpopulationsareclearlyresolvableintheMoxiGOIIoutput.Inthiscase,thecellsamplewaspre-treatedthrougha4hourincubationin20µMcamptothecin,acelltoxinknowntointerferewithDNAreplicationand,asresult,induceapoptosis.Thatpopulationwasthenmixedwith50%heat-killedJurkatstogenerateanecroticpopulationformorecompletegraphicalrepresentationsofthepossibleapoptosisstates.Theresultingmixturereflectsthe46.6%late-stage/necroticcells(upper-right,mostlyheat-killed),the38%healthycells(lower-right,nostain),andthe14.7%early-stageapoptoticcells(top-left,camptothecin-treatmentinducedapoptosis).Figure2balsopresentsthequadrant-gateddataintabularandbar-chartformatforeasierinterpretation/displayoftheresults.Boththequadrantchartandtablechartcanbeuser-printedinBMPformattogeneratedocumentimages(aswasdoneforthisappnote).TheMoxiGOIIhasbuilt-infunctionalityforrapidlycomparingtheresultsoftwoassaysfrombothvisualandquantitativeperspectives.Figure2cprovidesanexampleofthecomparisonoftheAnnexinV+measurementsoftheJurkatcellswhenfirstexposedtocamptothecin(t=0hour)andafterprolonged(t=6.5

Figure2–a.)TypicalMoxiGOIIapoptosis(FITC–AnnexinV)scatterplot(FITCvs.PI)output for Jurkatcells treatedwithcamptothecin(20µM,4hours)mixedwith50%heat-killedcells.Thequadrantplotsshowsthethreeexpectedstatesfortheculture: Live, Early-Stage Apoptosis, and Late-Stage Apoptosis. b.) Data can bepresented in tabular/bar chart format for easy interpretation of states c.) Thesystemiscapableofgeneratingcomparison/overlaysofdatafortwotests.Shownis an overlay of the Annexin V fluorescence vs. cell size scatter plots for acamptothecin-treated(separatefromabove)Jurkatsampleatthet=0(grayscatterplot) and t=6.5 hour (colored scatter plot) time pointsd.) This data can also bedisplayedasFITCfluorescencehistogramsforthet=0hour(graycurve)andt=6.5hour(redcurve)samples.

G e m i n i B i o A p p l i c a t i o n N o t e 3 / 2 0 2 1

Rapid Apoptosis Monitoring Using Gemini’s Bio’s Moxi GO II and Apoptosis Kit

hour)camptothecinexposure.Specifically,Figure2cshowstheoverlayofthefluorescence(FITC-AnnexinV)vs.sizescatterplotsforthetwosampleswiththeinitialreading(t=0hour)showningrayandthecoloredscatterplotshowingthecellsafter6.5hourofcamptothecintreatment.Figure2dprovidesthecorrespondingfluorescence(FITC-AnnexinV)histogramoverlayforbothsamples(graycurveisintheinitialreading,redcurveisthe6.5hourpost-treatmentreading).ThedataherevisuallyshowstheclearincreaseinAnnexinV+cellpercentagesforthetreatedsampleaswellasthecorrespondingdecreaseinthemeancellsize,awell-definedcharacteristicofapoptoticcells.ThisdiscriminationofthecellsizeshiftisuniquelyenabledontheMoxiGOthroughthepreciseCoulterPrinciplesizingofthecells,afeaturenotavailableonanyotherflowcytometers.Becauseoftheease-of-use,rapidtestandoperationtime,andtheconvenienceoftheMoxiGOII,itisuniquelysuitedtocollectionoftime-coursedata.Figure3showstheapplicationoftheMoxiGOIItothemeasurementofthetime-courseofcamptothecin-inducedapoptosisthroughmeasurementsofAnnexinVexpressionandPI-basedviability.Thegreenmarkersrepresentthecalculated(AnnexinV+%minusPI+%)earlystageapoptosispercentages(AnnexinV+/PI-%)ofthecamptothecin-treatedsamplevs.thenegativecontrol(treatedwithDMSO-only)asafunctionoftheelapseddrug-exposuretime.Plottingthedatainthismannerallowsforcurve-fitting(sigmoid,dashedgreenline)toextracttheapoptosisactivationkinetics(base=5.9%,max=34.2%,rate=35.7%/hr,halftime=4hr)ofthesecellsinresponsetotheappliedcamptothecindose.TheindividualPI-basedviabilitymeasurements(Figure3inset)wereusedtoquantifythelate-stageapoptosis/necroticcellpercentagesaswellastoconfirmtheviabilityofbothsamplesoverthecourseoftheAnnexinVmeasurements.TheconvenienceoftheMoxiGOIIreadilyenablesthis

Figure3 -Time-courseofapoptosis induction Jurkatcells. Apoptosis(FITC-AnnexinV)wasmeasuredontheMoxiGOIIateightdiscrete timepoints for aCamptothecin-treated (30µM) sample anda control (DMSOonly) sample. (Inset) Shows correspondingviabilitymeasurement(PI-permeability)ofeachsampleoverthesametimecourse.Thedataforthetreatedsamplewasfitwithasigmoidinordertoquantifythecamptothecinactivationkinetics.

G e m i n i B i o A p p l i c a t i o n N o t e 3 / 2 0 2 1

Rapid Apoptosis Monitoring Using Gemini’s Bio’s Moxi GO II and Apoptosis Kit

typeofprolongedtime-basedanalysis,anapproachthatcanbeprohibitivelyexpensive(bothcostandtime)inlabsthatarenototherwiseequippedwithcontinuallyoperatedflowinstrumentation.ConclusionsThedatainthisstudyshowstheversatilityoftheMoxiGOIIinmonitoringandquantifyingapoptosis.SpecificallyresearcherscanmeasuretheapoptosisstateofcellsbymonitoringthetranslocationofPSfromtheinnertotheouterleafletoftheplasmamembraneusingAnnexinV.CellviabilitycanbecorrespondinglyassessedusingPI.Furthermore,thesystemprovidesthecapabilityformonitoringthe characteristic size shift of the apoptotic cells (through precise Coulter Principle-basedmeasurements).OneofthemostpowerfulfeaturesoftheMoxiGOIIinstrumentistheease-of-useandconvenienceinthecollectionofdata.Withanabilitytoruntestswithouttheneedforsystemwarm-up,maintenance, or shutdownprocedures, theMoxi GO II is ideally suited to time courseexperiments, includingthemonitoringofapoptosis. Finally,astheMoxiGOIItouchscreenGUIisdesignedtomakeeventhemostcomplexflowanalysisaccessibletoresearchers,regardlessoftheirflow expertise. These features shouldmake theMoxi GO II indispensable in any lab performingapoptosismeasurementsorothercell-basedflowcytometrytechniques.

G e m i n i B i o A p p l i c a t i o n N o t e 3 / 2 0 2 1

Rapid Apoptosis Monitoring Using Gemini’s Bio’s Moxi GO II and Apoptosis Kit

References1. TeruKamogashira,ChisatoFujimoto,andTatsuyaYamasoba,“ReactiveOxygenSpecies,Apoptosis,

andMitochondrialDysfunctioninHearingLoss,”BioMedResearchInternational,v.2015,ArticleID617207,7pages,2015

2. PeterM.KangandSeigoIzumo,“ApoptosisandHeartFailure–ACriticalReviewoftheLiterature,”CirculationResearch,2000,v86,1107-1113

3. ScottW.LoweandAthenaW.Lin,“ApoptosisinCancer,”,Carcinogenesis,2000,v21(3),485-495.4. MarkP.Mattson,“ApoptosisinNeurodegenerativeDisorders,”Nature,Nov.2000,v1,120-129.5. B.Favaloro,N.Allocati,V.Graziano,C.DiIio,andV.DeLaurenzi,“RoleofApoptosisinDisease,”Aging,

May2012,v4(5),330-349.

MethodsCellCultureJurkatE6-1(ATCC)werecultured(37°C,5%CO2)inRPMI-1640supplementedwith10%FBS,1mMSodium Pyruvate, and 10mM HEPES (all Life Tech.). For apoptosis induction, 3µL of 10mMcamptothecin(Tocris)stock(inDMSO)wasaddedpermlofculturemedia(30µMfinalcamptothecinconcentration).Forthenegativecontrol,DMSOanequivalentvolumeofDMSOwassubstitutedforthecamptothecintreatment.Forgenerationofanecroticcellpopulation,healthyJurkatcellswereheat-killedbyplacinga15mlcentrifugevialswithcellsincubationina60°Cwaterbathfor10min+.The necrotic populationwasmixed 1:1 with a Camptothecin treated (20µM, 4hr) population togeneratea“threequadrant”Apoptosisexample.

FITC-AnnexinVAssayDual-labeldata(Figure2aand2b)werelabeledwithFITC-AnnexinV(GeminiBio’sVMoxiGoII488validatedkit,Catalog#MXA701)andPropidiumIodide(2µg/ml)followingGeminiBio’s“MoxiGO–EarlyStageApoptosisMonitoringwithAnnexinV”protocol(below).Afterpreparation,cellswererunontheMoxiGOIIsystem(GeminiBioCat#MXG112).Forthetimecoursedataandoverlaydataa1PMT/channelversionoftheMoxiGOIIsystemwasused.OtherthanthePMTconfiguration,thesystemhasthesamearchitectureastheMoxiGOIIsoperformancewouldbeequivalent.Forthosesamples,aseparateprepwassimultaneouslystainedwithPItogetthecorrespondingviabilitydata.Forthetime-coursedata,cellswereassayedontheMoxiGO(1PMTversion)ateightdiscretetimepointsovera~9hourperiod. AnnexinVandPImeasurementsweremadeateachtimepointforboththecamptothecin-treatedandnegativecontrolsamples.Screenshots&DataAnalysisScreenshotswereallgenerateddirectlyfromtheusingthebuilt-insystemscreenshotfunctionality(exportedscreenshotsappearonsystemdriveasBMPs).Datacomparisons/overlayswereallperformedon-unitusingthebuilt-insystemfunctionalityforcomparingsavedtests.FinalimagecroppingandarrangementwasperformedusingPhotoshop(Adobe)andIllustrator(Adobe).TimecoursedatawasextractedbyloadingtheMoxiGOFCSfilesintoFlowJo10.2(TreeStar),runningonMacOSX10.11.SummarydataforapoptosisandviabilitypercentageswereloadedintotheIGORPro(v6.37,Wavemetrics,Inc)analysispackageforcurve-fittingandfinalgraphgeneration.

G e m i n i B i o A p p l i c a t i o n P r o t o c o l 8 / 2 0 1 7

MoxiGO–EarlyStageApoptosisMonitoringwithAnnexinV

Instrument/Cassettes:

§ MoxiGOIINextGenerationFlowCytometer(GeminiBio-Products,Cat#MXG102)§ TypeS+Cassettes(GeminiBio-Products,Cat#MXC030/MXC032)

Reagents/Components:• MoxiCyteApoptosisKit(GeminiBio-Products,Cat#MXA701).Containing:

o Reagent#1:FITC–AnnexinVo Reagent#2:PropidiumIodideo Reagent#3:AnnexinV–BindingBuffer

Protocol:Notes:

• Forcomparisonpurposes,itcanbeusefultogenerateapositivecontrolbyinducingapoptosiswithapharmacologicalagent(e.g.30μMCamptothecintreated,4+hours,37°CforJurkatcells).

• Processasampleofhealthy,untreated,cellsforuseasanegativecontrol.1. Isolatecellstoasingle-cellsuspension.Note:Ifnecessary,useaprotease(e.g.Accutase,GemBio

Cat#400-158)and/orpipettetriturationtobreakaparttheclusters.2. (Optional)Forimprovedstainingresults,particularlywithadherentcells,pre-Washcells1x

(300xg,5min)withPBSorequivalent.3. PelletCells(300xg,5min)Re-suspendpelletto~1x106cells/mlinReagent#3:AnnexinV

BindingBuffer.4. Aliquot100μlofcellstoamicrocentrifugetube(~1x105totalcells).Mixwellbefore

aliquoting.5. Add5µLofReagent#1:FITC-AnnexinVconjugate.Note:While5µLshouldworkformostcell

samples,itmaybenecessarytotitratetheReagent#2volumetooptimizethesignal.6. Add5μlofReagent#2:PropidiumIodide(PI)7. Gentlyvortex(3-4setting)thecellsandincubatefor15minutesatroomtemperature(25°C),

protectedfromlight.8. Add390µLofReagent#3:AnnexinVBindingBuffertoalltubes.9. RunonMoxiGOIIusingthe“Apoptosis(AnnexinV-FITC&PI)”appwithin15minutesof

staining.Protectfromlight.Notes:a. Thiskitwasdesignedtobeusedwiththe646nm/LPfilterinstalledintheback(PMT2)

slotoftheMoxiGOII.Usingthe561nm/LPfilterwillrequirecompensatingforspillover(FITCintoPMT2)andpossiblyloweringthePIconcentrationsothatitisnottoobright.

b. Adjustsizegatestodefinethecellpopulation.c. Touch“Next”viewPMTvsPMTdisplayoftheFITCAnnexin(PMT1)vs.PI(PMT2)

fluorescence.Adjustthegatemarkerstoidentifytherelevantcellsub-populations/d. Oncegated,touch“Summary”forabarchart/tablesummaryviewofthedata.

G e m i n i B i o A p p l i c a t i o n P r o t o c o l 8 / 2 0 1 7

MoxiGO–EarlyStageApoptosisMonitoringwithAnnexinV

http://www.gembio.com