DetectionOfCellViabilityAnd/OrApoptosisByFlowCytometry(FACS)SP,September2005Viability,apoptosis,necrosis,deathViablecellsarecellsthatwhenallowedtocontinuebeyondthetimepointofexaminationwillstayalive.Besidesliveandhealthycells,cellsinearlystagesofapoptosismaybeconsideredviableasearlyapoptosisisbelievedtobereversibleiftheconditionsinducingapoptosisareremoved.Conditionsinducingapoptosisincludewithdrawaloforexposuretocertainfactors(e.g.,withdrawalofNGF,orexposuretoTNF-α).Theseconditionsarepresentedinvivo normally (e.g.,duringmaturationoflymphocytes)or abnormally (e.g.,duringviralinfections),orinvitrowithourchoice(e.g.,additionofcampotheticinchemicaltocellculture)orwithusunawareoforineffectiveagainstthem(e.g.,lackofgrowthfactorsduringhandling).Apoptosisultimatelyleadstodeathandthiscantakefromsomeminutestomanyhours.The earlystagesofapoptosisarecharacterizedbychangestomitochondrialmembranepotential andcellmembraneasymmetry(butnotincreasedcellpermeability).LaterstagesarecharacterizedbyDNAfragmentationandlossofcellmembranepermeability.Ingeneral,apoptoticcellsshrink(andcanbreakupintosmallerapoptoticbodies)andhavecharacteristicnuclearchangesthatarevisibleunderanelectronmicroscope.Necrosis,unlikeapoptosis,isnotastep-wise,controlledphenomenon.Itiscausedbyphysicaldisruptionofthecellbyphysical(e.g.,heat)orchemical(e.g.,lowpH)means,microbialtoxins(e.g.,bycausingincreasedcellpermeability),etc.Earlynecroticcellshaverelatively-normallookingnucleibuttheircellmembranes(andorganellarmembranes)arefragmented.Withtime,organellar(includingnuclear)disruptiontakesplaceandthecellsgenerallyswell,eventuallybursting(andreleasinginflammationinducers).Apoptoticcellsinthelaststagescannecrosetoo.Cytometriccharacteristicsofnon-viablecellsAsampleofcellsbeingexaminedbyflowcytometry(e.g.,forexpressionofacertainantigen)willhavesomenon-viablecells(arisingbecauseofapoptosisand/ornecrosis-seeaboveforreasons).Unlessoneisspecificallymeasuringapoptosis,thesenon-viablecellsshouldbeexcludedfromexaminationbygating.Thisisbecause,forexample,theymaynon-specificallybindafluorescentsecondaryantibody,theantigenbeingdetectedmayhavebeendegraded,etc.Non-viablecellstendtohavereducedFSC(canbehighincaseofswollennecroticcells)buthigherSSCvalues.Becauseofmembranepermeabilityincreases,DNA(anddependingonthereagent,RNAtoo)ofdeadcells,necrosingcellsandcellsinlateapoptosiswillbindreagentssuchaspropidiumiodide(PI ;MW668)and7-actinomycinD(7-AAD;MW1270)tofluorescemore.Becauseoflossofcellmembraneasymmetry(forexample,leadingtotheexposureofphosphatidylserinebecauseofitsincreasedpresenceontheouterleaflet),deadcells,necrosingcellsandcellsinearlystagesofapoptosiscanbedetectedbybindingofannexinV(aphosphatidylserine-recognizingprotein).

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