METHOD:

Principle:

Apoptoticcells,probablyduetoachangeinmembranepermeABIlity,takeupsome7-AADandbecome7-AADdimcomparedtolivecellswhichremain7-AAD-.Lateapoptoticornecroticcellswhichhavelostmembraneintegrityappear7-AADbright.

Reference:SchmidI,UittenbogaartCH,KeldB,andGiorgiJV:Arapidmethodformeasuringapoptosisanddual-colorimmunofluorescencebysinglelaserflowcytometry.JImmunolMeth170:145-157,1994.

Stainingfordetectionofapoptosis

1x106PBSAz-washedcellsfromasinglecellsUSPensionarepelletedina12X75mmculturetube.Thepelletisresuspendedin1mlof1XPBSAzcontaining20micrograms/mlof7-AADandthesuspensionismixedgently.Afterapproximately20minofincubationat4^°Cinthedark,thesamplescanberunontheflowcytometerintheirstainingsolution.Theyshouldbekeptcoldduringacquisitionandshouldberunwithin1haftertheadditionof7-AAD,otherwisecellsshouldbefixed.

Note:itispossIBLetofixcellsstainedwith7-AADfordetectionofapoptosiswitha1%formaldehydesolutioncontaining20micrograms/mlofactinomycinD(AD,C₁,e.g.,BoehringerMannheim)forbiohazardconsiderationsand/ortopreservesamplesforlateranalysisontheflowcytometer.Forprocedureseeattachedprotocol.

Thisstainingprocedureallowsthecorrelationofapoptosiswithsingleordual-colorphenotypeandtheuseofFITCand/orPEconjugatedmonoclonalantibodies.Surfaceantigenstainingisdoneaccordingtostandardmethodsbeforestainingforapoptosis.Then,theapoptosisassayisperformedasdescribedabove.

DirectStainingProcedure

1.Resuspendcellpelletfirstin50microlitersofHABforapproximately1min,thenadd50microlitersofPBSAzandtheappropriateamountofFluorochrome-conjugatedmonoclonalantibody(mAb).

2.Vortexbrieflyandincubatefor15minat4^°Cinthedark.

3.Washoncewith2mlsofPBSAzbycentrifugationat250gfor5minutes.

4.Proceedwithstainingforapoptosisasdescribedabove.

IndirectStainingProcedure

1-3Processsamplesasaboveusingaworkingdilutionofunlabelledantibody.

4.Resuspendcellpelletfirstin50microlitersofHABforapproximately1min,thenadd50microlitersofaworkingdilutionofthefluorochrome-conjugatedsecondantibody.

5.Vortexbrieflyandincubatefor20minat4^°Cinthedark.

6.Washoncewith2mlsofPBSAzbycentrifugationat250gfor5min.

7.Proceedwithstainingforapoptosisasdescribedabove.

Note:donotuseHABforstainingofimmunoglobulinchains.Wheneveravailable,useamonoclonalantibodyand/orareagentdirectlyconjugatedtoafluorochrometominimizeunspecificbinding.Alwaysuseisotypiccontrolsofthesameheavychainclassasyourrelevantantibodyfordeterminationofbackgroundstaining.

ProtocolfortheuseofactinomycinD(AD)onsamplesthatwerestainedwith7-AADforapoptosisandfixedinformaldehyde.

I.Materials:

1.ActinomycinD(AD,C₁,e.g.,BoehringerMannheim,IN)

2.1XPBS

3.Sonicator

4.Formaldehydesolution(seeprotocolforpreparationof2%stockformaldehydesolution)

II.PreparationofADstocksolution(1mg/ml):

To1mgofADpowderadd:

50microlitersoficecoldabsoluteETOH,vortex

950microlitersof1XPBS

Sonicatetheresultingsolutionfor10minat4^°C;keepthesolutionovernightintherefrigeratorat4^°C,protectedfromlightbeforeusingit.

Storesolutionat4^°Cprotectedfromlight.

Workingdilutionis20micrograms/ml.

III.Method:

Cellsarefirstincubatedwith7-AADforatleast20min,thentheyarespundownonceandimmediatelya1%formaldehydesolutioncontaining20microliters/mlofAD(F/AD)isaddedtothecellpellet.Cellshavetobestoredinthecoldprotectedfromlightandcanbeanalyzedapproximately30minaftertheadditionoftheF/ADsolution.CellsarerunontheflowcytometerintheF/ADsolution.Wehavestoredsamplesupto3dayswithoutanylossintheabilitytodiscriminateearlyapoptoticfromlivecellsandlateapoptoticornecroticcells.

INFORMATIONANDREFERENCESONDETECTIONOFAPOPTOTICCELLSBYFLOWCYTOMETRY

ComparedtotheclassicmethodsofDNAladderformationbygelelectrophoresisandofmorphologicexaminationbyelectronmicroscopyfordeterminationofapoptosis,flowcytometrypermitsrapidandquantitativemeasurementsonapoptoticcells.Manydifferentflowcytometricmethodsfortheassessmentofapoptosisincellshavebeendescribed(forareviewsee1);mostofthesemethodsmeasureapoptoticchangesincellsbystainingwithvariousDNAdyes,however,techniquesusingtheterminaldeoxynucleotidyltransferaseornicktranslationassayshavealsobeendeveloped(3).Someofthesestainingmethodsutilizeunfixedcells(2,4,5,7,8,9,10,11).Duetothefragilityofcellsundergoingprogrammedcelldeath,rapidmethodsthatmaintaincellsascloseaspossibletotheirnaturalstatemightbeexpectedtoprovidethemostreliableresults.Thecurrentrapid7-AADstainingmethodusesunfixedcellsandthuspermitsthedetectionofchangesinlightscatterparametersandtheircorrelationwithotherindicatorsofprogrammedcelldeath.However,onedrawbackofusinganylivestainingmethodformeasuringapoptosisisthevariabilityofdyeuptakeindifferentcellsanditspossiblechangethroughcertaintreatmentconditions.FurThermore,reagentswhichaffectmembranepermeability(e.g.calciumionophores)cannotbeusedwiththistechnique.

ManystainingmethodsforflowcytometryuseeitherfixedcellsortreatcellswithahypotonicsolutiontopermitDNAstainingbynon-vitaldyes.TheapoptoticcellswithdegradedDNAappearascellswithhypodiploidDNAcontentandarerepresentedinso-called"sub-G1"peaksonDNAhistograms(6,12).Telfordet.al.(12)showedonmurinethymocytesthatmanydifferentDNAdyesproducesimilardistributionsof"sub-G1"peaksirrespectiveoftheirbindingmodewhenthesamecellfixationmethodwasused.However,whenwetriedtoapplythehypotonicPImethodbyNicolettietal.(6)tohumanthymocytesourresultsindicatedthatuseofdissimilarsamplepreparationmethods(e.g.livestainingwithHO342vs.hypotonicPI)canleadtodramaticdifferencesintheabilitytodetectapoptoticcells.Undercertainconditions,thetreatmentofhumanthymocyteswithhypotoniccitratesolutioncontainingdetergentmaynotpermitleakageofthelowmolecularweightDNAoutofapoptoticcells;thisleakagehasbeenproposedasapre-requisitefortheformationof"sub-G1"peaks(1).Inmurinethymocytes,however,thehypotonicPIstainingmethodproducesacleardistinctionbetweenliveandapoptoticcells(6).

Recently,ithasbeenpostulatedthatthestabilityandthekineticsofintermediatecelldeathstagesmaydeterminetheresistanceofaparticularcelltypetoDNAdegradation.Consequently,thevisibilityof"sub-G1"peaksbyflowcytometryandalsotheformationofcharacteristicDNAladdersonagarosegelsmightbeafunctionofindividualcellularhomeostasis.Theabsenceofa"sub-G1"peakonaDNAhistogramshouldnotconstituteproofofnoapoptosis(1).Conversely,ithasalsobeenstatedthatthemereappearanceofahypodiploidDNApeakshouldnotbetakenasdefiniteevidenceforthepresenceofanapoptoticcellpopulationwithoutothersupportinginformation(7).Flowcytometricfindingsinaparticularcelltypeexposedtoacertainstimulusmustthereforebealwaysverifiedwithothernon-flowcytometricmethods.Recently,rapidflowcytometricstainingmethodsthatuseAnnexinVfordetectionofphosphatidylserineexposureonthecellsurfaceasaMarkerofapoptosishavebecomecommerciallyavailable.Forthisstainingmethoditisessentialtoaddadeadcelldiscriminationdyelikepropidiumiodideor7-amino-actinomycinDtothestainedcells,becauselateapoptoticornecroticcellsalsoexpressphosphatidylserineandhavetobedistinguishedfromtheearlyapoptoticcellsbyfluorescence(13).ThenewestflowcytometricassaysmeasureCaspase-3activity,anearlymarkerofcellsundergoingapoptosisandkitsforperformingthisassaysarecommerciallyavailable(14).

Pleasekeepinmindthatnotallmethodsfordetectionofapoptosisincellsmaybeequallysensitiveandtechniquesmustbeassessedcriticallywithrespecttotheirapplicabilitytoaparticularcelltypeorsystem.