AndreaCossarizzaandStefanoSalvioliDepartmentofBiomedicalSciencesUniversityofModenaSchoolofMedicineviaCampi287,41100Modena,Italyphone+3959428.613fax+3959428.623Email:cossariz@unimo.it

Introduction

ThemitochondrialrespiratorychainproducesenergywhichisstoredasanelectRochemicalgrADIentwhichconsistsofatransmembraneelectricalpotential,negativeinsideofabout180-200mV,andaprotongradientofabout1unit;thisenergyisthenabletodrivethesynthesisofATP,acrucialmoleculeforaconsistentvarietyofintracellularprocesses.Severalmembranepermeablelipophiliccations,accumulatedbylivingcells,organellesandliposomesexhibitinganegativeinteriormembranepotential,havebeenusedtostudyDy.Suchprobesincludethosewhichexhibitopticalandfluorescenceactivityafteraccumulationintoenergizedsystems,suchas3,3"-diehexiloxadicarbocyanineiodide[DiOC₆(3)],nonylacridineorange(NAO),safranineO,rhodamine-123(Rh123)etc.,radiolabelledprobes,(i.e.,[³H]methyltriphenyl-phosphonium,etc.)andunlabelledprobesusedwithspecificelectrodes[i.e.,tetraphenyl-phosphoniumion(TPP+)etc.].ThesesystemshaveseveralpossIBLedisadvantages,includingthe:a)timerequiredtoachieveequilibriumdistributionofamitochondrialmembraneprobe;b)degreeofpassive(unspecific)bindingofprobestoamembranecomponent,suchasinthecaseofNAO,whichdetectsmitochondrialmassasitbindstocardiolipin(9),orRh123,whichhasseveralenergy-independentbindingsites(10),orDiOC₆(3)which,notwithstandingitshighcapacitytobindothermembranesthanthoseofmitochondriaanditslowsensitivitytoagentscapableofdepolarizesuchorganelles(11,12),hasbeenwidelyusedinthelastyearsforstudiesonDy;c)toxiceffectsofprobesonmitochondrialfunctionalintegrity;d)samplingprocedures;e)interferencefromlightscatteringchangesandfromabsorptionchangesofmitochondrialcomponents;f)requirementoflargeamountsofBIOLOGicalmaterials.TPPelectrodeaffordsaneasyandprecisetooltomeasureDyduetothe:i)lowinterferencebetweenboundTPP+andthemembrane;andii)lackofresponsesoftheelectrodetospeciesdifferentfromTPP+.However,thismethodrequiresdiscreteamountsofbiologicalsamplesanduptakeofthislipophiliccationbyintactmammaliancellsisindeedaslowprocess.

TodetectvariationsinDyatthesinglecelloratthesingleorganellelevel,afewyearsagowehavedevelopedanewcytofluorimetric(FCM)techniquebyusingthelipophiliccation5,5",6,6"-tetrachloro-1,1",3,3"-tetraethylbenzimidazolcarbocyanineiodide(JC-1)(13-15).JC-1ismoreadvantageousoverrhodaminesandothercarbocyanines,capableofenteringselectivelyintomitochondria,sinceitchangesreversiblyitscolorfromgreentoorangeasmembranepotentialsincrease(overvaluesofabout80-100mV).ThispropertyisduetothereversibleformationofJC-1aggregatesuponmembranepolarizationthatcausesshiftsinemittedlightfrom530nm(i.e.,emissionofJC-1monomericform)to590nm(i.e.,emissionofJ-aggregate)whenexcitedat490nm;thecolorofthedyechangesreversiblyfromgreentogreenishorangeasthemitochondrialmembranebecomesmorepolarized(16-18).Bothcolorscanbedetectedusingthefilterscommonlymountedinallflowcytometers,sothatgreenemissioncanbeanalyzedinfluorescencechannel1(FL1)andgreenishorangeemissioninchannel2(FL2).ThemainadvantageoftheuseofJC-1isthatitcanbebothqualitative,consideringtheshiftfromgreentoorangefluorescenceemission,andquantitative,consideringthepurefluorescenceintensity,whichcanbedetectedinbothFL1andFL2channels.

Clearly,Dyhasbeenpreviouslystudiedbyflowcytometry,mostlybyevaluatingthechangesinfluorescenceintensityofcellsstainedwithdifferent,cationicdyes.ResearchersusedfirstRh123(19-21),thenothermoleculessuchasDiOC₆(3)(22).Typically,thesignalcomingfromcellswhosemitochondriahadalowpotentialwasmuchlowerthanthatofcontrolsamples,andinaclassicalhistogramdepolarizedpopulationsgototheleft.However,aftertheshifttotheleftthepeaks(i.e.thatofcontrolsandtreatedcells)arenotalwaysperfectlyseparate,theoperatorhastodecide"byeye"wherethepopulationofcellswithdepolarizedmitochondriabegins.Thesetwofluorescentprobeshavethisandotherproblems.Rh123bindingtomitochondriaisdifficulttocalculatewhenthecellhasacertainmitochondrialheterogeneitydue,forexample,toahighnumberofmatureorimmatureorganelles,asoccursinacontinuouslygrowingcellline.Moreover,differentmitochondrialbindingsitesforRh123exist,i.e.siteswhicharefreelyaccessiblewhatevertheenergystatusofthemitochondriaandsiteswhicharehiddenintheenergizedstateandfreelyaccessibleinthedeenergizedformoftheorganelle.Thishasbeenattributedtodifferentmaturativestatesoftheorganelles.Thus,inasinglecell,organellescanhavedifferentRh123bindingsiteswithconsequentdifferentfluorescenceemissions.Asaresult,itisverydifficulttoascertainwhetherornotmitochondriabindRh123inanenergy-dependentorenergy-independentmanner.However,theprobeisperfectwhenusedinassociationwithpropidiumiodide,asthiscombinationallowsaclearandelegantdistinctionbetweendeadandlivingcells(4).

DiOC₆(3)ismorereliableforanalysisofplasmamembranepotentialratherthanforstudiesonDY.Indeed,thefirstapplicationofthisprobeinFCMwasfortheanalysisofplasmamembranepotential(23).Afterthis,DiOC₆(3)wasusedinisolatedmitochondriatodetectDychanges(24).Anycationicmoleculegoestonegativesites,andcanbereleasedwhenthenegativechargedecreases.Ifthatmoleculeisfluorescent,thesignaldecreaseswhenthemembranepotentialoftheorganelleislost.Fluorescentmoleculespresentinintactcellshaveadifferentbehaviour.Inourhands,DiOC₆(3)reactedproperlywhenU937cellsweretreatedwithFCCP,butsuchbehaviourwasnotobservedincellstreatedwithvalinomycin.Moreover,whencellswerekeptinthepresenceofplasmamembranedepolarizingagentssuchasouabainorhighdosesofextracellularK+,aconsistentdecreaseinDiOC₆(3)fluorescencewasnoted,indicatingaconsistentsensitivityoftheprobeforplasmamembrane(12).Thisbehaviourwasnottotallyunexpected,asitisknownthatthisprobecanbindseveralmembranesotherthanmitochondria,asalsoreportedintheHandbookofFluorescentProbesandResearchChemicals(editedbytheCompanythatproducesandsellsthisreagent,i.e.Molecularprobes,Eugene,OR,USA).Thus,usingthisprobe,itisverydifficult,ifnotimpossible,todistinguishbetweendepolarizationofplasmamembraneorchangesinDYinseveralphysiologicalorpathologicalconditions,suchasapoptosis,whenbotheventscantakeplace.

2.PROTOCOL

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JC-1staining
2.1MaterialsJC-1fluorescentprobe,plastictubesforFACSanalysis,completemedium,i.e.RPMIaddedwith10%fetalcalfserum,PBS.2.2.Methodology1.Harvestcells(atleast2x10⁵)fromexperimentalsamples,bringtotalvolumeupto1mLoffreshcompletemedium.

2.StaincellsUSPensionwith2.5mg/mLJC-1.Shakecellsuspensionuntilthedyeiswelldissolved,givingauniformred-violetcolor.Todothis,itisalsopossibletovortexvigorouslythesuspensionimmediatelyaftertheadditionoftheprobe.

3.Keepthesamplesinadarkplaceatroomtemperaturefor15-20minutes.Thedurationofthestainingdependsuponthecelltype,butinourhandsallthecellsused(lymphocytes,celllinesofdifferentorigin,fibroblasts,keratinocytes,hepatocytes,etc.)respondedquitewelltothetreatment.Washtwicecentrifugingat500gfor5minwithadoublevolumeofPBS.

4.Resuspendin0.3mLofPBS,thenanalyzeimmediatlywiththeflowcytometer,typicallyequippedwitha488nmargonlaser.Setthevalueofphotomultiplier(PMT)detectingthesignalinFL1atabout390V,andFL2PMTat320V;FL1-FL2compensationshouldbearound4.0%,whileFL2-FL1compensationaround10.6%.Thisishowevertheclassicalsettingoftheinstrumentweuseinourlaboratory,andithastobetakenintoaccountthat,aseachinstrumenthasadifferentsensitivity,adifferentsettingcanbenecessarytoobtainanoptimalsignal.Concerninginstruments,thestaininghasbeentestedonseveraldifferentapparatussuchasanExcel,fromCoulter(inBergen,Norway),anElite(Coulter)inParis,someFACSCAN,aFACSTARPlusandaFACSCalibur,fromBectonDickinson(inKrakow,Poland,orModenaandVenice,Italy),aBioradBriteandaPartec(inKrakowtoo),andtheyworkperfectlyaswell.Obviously,compensationshavetobesetinadifferentway.

3.COMMENTARY

3.1BackgroundinformationThetechniqueofJC-1staininghasbeendevelopedwiththeintenttodetectDYinintact,viablecells.ForthispurposeJC-1actsasaMarkerofmitochondrialactivity,sincetheformationofJ-aggregates,whichgiveredemission,isreversible.CellswithhighDYarethoseformingJ-aggregates,thusshowinghighredfluorescence.Ontheotherhand,cellswithlowDYarethoseinwhichJC-1maintains(orre-acquire)monomericform,thusshowingonlygreenfluorescence.NormallygreenfluorescenceofdepolarizedcellsisalittlebithigherthanthatofpolarizedonessimplybecauseofthepresenceofahigheramountofJC-1monomers.

Duringtheiruse,allreagentsmustbeatroomtemperatureandcarefullycheckedforpH(7.4),sincemitochondrialDYisverysensitivetoalterationsofbothparameters.

Stainingproceduremustbecarriedundernodirectintenselightandincubationinthedark,becausethelightsensitivityofJC-1.

AlwaysweargloveswhenhandlingJC-1.

3.2CriticalParameters

TheneedofhighDYfortheformationofJ-aggregatesmakesthisstainingnotsuitableforfixedsamples.Indeed,thistechniquehastobeconsideredasafunctionalitytest.Possibledisadvantagescomefromthewideemissionspectrumofthedye,whichoccupiestwofluorescencechannels,thusavoidingtheuseofotherprobesconjugatedwithFITC(e.g.monoclonalantibodies).ThecouplingwithprobesemittingindeepreddetectableinFL3channelistheoreticallypossible,buthasmanyproblemsincompensatingthedifferentfluorescences,dependingontheparticularemissionspectrumofeachprobe(quenchingphenomenon).Inparticular,usingpropidiumiodideforassessingcellviABIlityincellslabelledwithJC-1cancreateconsistentproblems.

OnlyrecentlycantheAuthorstestthestabilityoftheprobeinlivingcellsfixedafterthestaining.ThiswasdonebecausecellswereinfectedwithHIV-1,anditisstronglyrecommendedtofixsuchcellsbeforerunningthemintoaflowcytometer.Alightfixationwith0.5%formaldeide(fewminutesatroomtemperature)howeverdoesnotchangethefluorescencepattern.

3.3Trobleshooting

1.PresenceoffluorescentmoleculesotherthanJC-1inthesample:analyzefirstanonstainedsampleandsettheinstrumentonitsspontaneusfluorescence,thenanalyzethestainedsampleswiththesamesetting.Seealsopoint3.2.

2.Cellsarenotwellstained:increasetheamountofJC-1.Trytostainwithanincubationat37°Cinsteadatroomtemperature.

3.Cellaretoomuchstained:decreasetheamountofJC-1.LeavethecellstostayalittlebitlongerintheJC-1freePBSinordertoallowthedyetoreachtheappropiatedistributionequilibrium.

4.Fluorescencepatterntoomuchwidespread:seepoint3.4.DonotconsideranyeventwithaveryhighFL2fluorescence:veryoftentheyareJC-1aggregates.IncreaseFSCthresholdanddiscarddebriswithelectronicgating:thepresenceofstaineddebrisorbrokencellscanconstituteaconfoundingelementinthewholefluorescencepattern.

3.4AnticipatedresultsItisrecommendedtoperformeachexperimentusinga"positivecontrol"sample,inwhichmitochondriaofallcellshavebeendepolarizedinordertohaveacorrectsettingoftheinstrument.TreatingcellswithdrugsabletocollapseDY,suchastheK+ionophorvalinomycin(100nMormore)ortheprotontranslocatorcarbonylcyanidep-(trifluoromethoxy)phenylhydrazone(FCCP,250nM),resultsinadramaticchangeofthefluorescencedistributionthatindicateswheredepolarizedcellshavetogoandhelpsalotinsettingthecompensation.Forproblemsrelatedtointracellulartraffickinganddrugneutralization,valinomycinworksmuchbetterthanFCCP(andisalsolessexpensive).

Whenthesamplecontainsanheterogeneiccellpopulation,itispossibletoseedifferentfluorescencepatternsduetothevariablecontentinmembranesandmitochondriaofcellsubpopulations.Itistypicalthecaseofperipheralbloodmononuclearcells(PBMC),formedbylymphocytesandmonocytes,thefirstbeingsmallerandwithlessmitochondrialcontentthanthelatter.Accordingly,thefluorescencepatternofJC-1ofsuchsampleshowstwodistinctpeaks,onecorrespondingtolymphocytes,andthesecond,brighterinbothFL1andFL2,correspondingtomonocytes.

Anothergoodcontrolisthatofmitochondrialmass,thatcanbedonewithnonylacridineorange(NAO),thatbindsmitochondriaindependentlyoftheirenergizationstate,andwhosefluorescenceisdetectableinFL1.Typically,cellsareincubatedattheconcentrationof0.5-1x10⁶cells/mLwith10µMNAO(MolecularProbes)for10min.inthedarkatroomtemperature,washedtwiceincoldPBSandimmediatelyanalyzed.Theresultyouobtaingivesyouanideaofthemassofmitochondriapresentwithinacell,andallowsyoutobesurethatthechangesyouseewithapotential-sensitiveprobearenotdependentuponthesimplelossoforganelles.Itiseasytoimaginethatalsointhiscasemonocytesarebrighterthanlymphocytes.

3.5TimeconsiderationsTheprotocolofJC-1stainingdoesnotrequirealongtime(moreorless30minutes).Thedurationofthestainingprocedurecanobviouslyincrease,dependingonthenumberofsamplestobeanalyzed.

3.6Keyreferences

1.KroemerG.,ZamzaniN.,SusinS.A.Mitochondrialcontrolofapoptosis.Immunol.Today,18:44-51,1997.

2.SusinS.A.,ZamzamiN.,CastedoM.,DaugasE.,WangH.G.,GeleyS.,FassyF.,ReedJ.C.,KroemerG.Thecentralexecutionerofapoptosis:multipleconnectionsbetweenproteaseactivationandmitochondriainFas/APO-1/CD95-andceramide-inducedapoptosis.J.Exp.Med.,186:25-37,1997.

3.KroemerG.Theproto-oncogeneBcl-2anditsroleinregulatingapoptosis.NatureMed.,3:614-620,1997.

4.CossarizzaA.,KalashnikovaG.,GrassilliE.,ChiappelliF.,SalvioliS.,CapriM.,BarbieriD.,TroianoL.,MontiD.,FranceschiC.Mitochondrialmodificationsduringratthymocyteapoptosis:astudyatthesinglecelllevel.Exp.CellRes.,214:323-330,1994.

5.RichterC.,SchweizerM.,CossarizzaA.,FranceschiC.ControlofapoptosisbythecellularATPlevel.FEBSLett.,378:107-110,1996.

6.GormanA.M.,SamaliA.,McGowanA.J.,CotterT.G.Usefoflowcytometrytechniquesinstudyingmechanismsofapoptosisinleukemiccells.Cytometry,29:97-105,1997.

7.YangJ.,LiuX.,BhallaK.,KimC.N.,IbradoA.M.,CaiJ.,PengT.I.,JonesD.P.,WangX.Preventionofapoptosisbybcl-2:releaseofcytochromecfrommitochondriablocked.Science,275:1129-1132,1997.

8.DeMariaR.,LentiL.,MalisanF.,d"AgostinoF.,TomassiniB.,ZeunerA.,RippoM.R.,TestiR.RequirementforGD3gangliosideinCD95-andceramide-inducedapoptosis.Science,277:1652-1654,1997.

9.MaftahA.,PetitJ.M.,RatinaudM.H.A.J.,R.10-Nnonyl-acridineorange:afluorescentprobewhichstainsmitochondriaindependentlyoftheirenergeticstate.Biochem.Biophys.Res.Commun.,164:185-190,1989.

10.Lopez-MediavillaC.,OrfaoA.,GonzalesM.,MedinaJ.M.Identificationbyflowcytometryoftwodistinctrhodamine-123-stainedmitochondrialpopulationsinratliver.FEBSLett.,254:115-120,1989.

11.TerasakiM.,SongJ.,WongJ.R.,WeissM.J.,ChenB.L.Localizationofendoplasmicreticuluminlivingandglutaraldehyde-fixedcellswithfluorescentdyes.Cell,38:101-108,1984.

12.SalvioliS.,ArdizzoniA.,FranceschiC.,CossarizzaA.JC-1,butnotDiOC₆(3)orrhodamine123,isareliablefluorescentprobetoassessDYinintactcells.Implicationsforstudiesonmitochondrialfunctionalityduringapoptosis.FEBSLett.,411:77-82,1997.

13.CossarizzaA.,BaccaraniContriM.,KalashnikovaG.,FranceschiC.AnewmethodforthecytofluorimetricanalysisofmitochondrialmembranepotentialusingtheJ-aggregateforminglipophiliccation5,5",6,6"-tetrachloro-1,1",3,3"-tetraethylbenzimidazolcarbocyanineiodide(JC-1).Biochem.Biophys.Res.Commun.,197:40-45,1993.

14.CossarizzaA.,SalvioliS.,FranceschiniM.G.,KalashnikovaG.,BarbieriD.,MontiD.,GrassilliE.,TropeaF.,TroianoL.,FranceschiC.Mitochondriaandapoptosis:acytofluorimetricapproach.Fund.Clin.Immunol.,3:67-68,1995.

15.CossarizzaA.,CeccarelliD.,MasiniA.Functionalheterogeneityofisolatedmitochondrialpopulationrevealedbycytofluorimetricanalysisatthesingleorganellelevel.Exp.CellRes.,222:84-94,1996.

16.HadaH.,HondaC.,TanemuraH.SpectroscopicstudyontheJ-aggregateofcyaninedyes.I.SpectralchangesofUVbandsconcernedwithJ-aggregateformation.Photogr.Sci.Eng.,21:83-91,1977.

17.ReersM.,SmithT.W.,ChenL.B.J-aggregateformationofacarbocyanineasaquantitativefluorescentindicatorofmembranepotential.Biochemistry,30:4480-4486,1991.

18.SmileyS.T.,ReersM.,Mottola-HartshornC.,LinM.,ChenA.,SmithT.W.,SteeleG.D.,ChenL.B.IntracellularheterogeneityinmitochondrialmembranepotentialrevealedbyaJ-aggregate-forminglipophiliccationJC-1.Proc.Natl.Acad.Sci.USA,88:3671-3675,1991.

19.JohnsonL.V.,WalshM.L.,BockusB.J.,ChenL.B.Monitoringofrelativemitochondrialmembranepotentialinlivingcellsbyfluorescencemicroscopy.J.CellBiol.,88:526-535,1981.

20.GoldsteinS.,KorczackL.B.Statusofmitochondriainlivinghumanfibroblastsduringgrowthandsenescenceinvitro:useofthelaserdyerhodamine123.J.CellBiol.,91:392-398,1981.

21.DarzynkiewiczZ.,Staiano-CoicoL.,MelamedM.R.Increasedmitochondrialuptakeofrhodamine123duringlymphocytestimulation.Proc.Natl.Acad.Sci.USA,78:2383-2387,1981.

22.PetitP.X.,LecoeurH.,ZornE.,DauguetC.,MignotteB.,GougeonM.-L.Alterationsinmitochondrialstructureandfunctionareearlyeventsofdexamethasone-inducedthymocyteapoptosis.J.CellBiol.,130:157-167,1995.

23.JenssenH.-L.,RedmannK.,MixE.Flowcytometricestimationoftransmembranepotentialofmacrophages-Acomparisonwithmicroelectrodemeasurements.Cytometry,7:339-346,1986.

24.PetitP.X.,O"ConnorD.,GrunwaldD.,BrownS.C.Analysisofthemembranepotentialofrat-andmouse-livermitochondriabyflowcytometryandpossibleapplications.Eur.J.Biochem.,194:389-397,1990.

Appendix1:Stocksolution:

JC-1isdissolvedinN,N’-dimethylformamide(Sigma-Aldrich,cat.n.D8654)attheconcentrationof2.5mg/ml.

Itisstoredat-20°C.Lightsensitive.

Appendix2:Reagents

JC-1MolecularProbes,Eugene,OR,USA

catalogNo.:T-3168

Note:colturemedium,salinesolutionsandwashingbuffersaredependingonthecelltypewhichisusedfortheexperimentalprocedure(PBMC,fibroblasts,hepatocytes,etc.).Forbloodwhitecells,RPMI1640with10%heatinactivatedfoetalcalfserum,100IU/mlpenicillin,100mg/mlstreptomycin,2mML-glutamineisnormallyusedascompletecolturemedium.

Appendix3:Equipment

FlowCabinetTC60	Gelaire
FlowCytometerFACScan	BectonDickinson
IncubatorCO2-AUTO-ZERO	Heraeus
CentrifugeMinifugeRF	Heraeus
PipetmanP20,P200,P1000	Gilson
VortexVibrofixVF1Electronic	Janke&Kunkel-IkaLabortechnik

Appendix4:Glossary

Mitochondrialmembranepotential(Dy)isgeneratedbymitochondrialelectrontransportchain,whichdrivesaprotonflowfrommatrixthroughinnermitochondrialmembranetocytoplasm,thuscreatinganelectrochemicalgradient.ThisgradientisinturnresponsiblefortheformationofATPmoleculesbyF₀-F₁ATPsynthase.ForthisreasonDyisanimportantparameterformitochondrialfunctionalityandanindirectevidenceofenergystatusofthecell.

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