流式细胞仪测定线粒体膜电位的操作方法

JC-1MitochondrialMembranePotentialAssayKitItemNo.100091723GENERALINFORMATIONTABLEOFCONTENTSGENERALINFORMATION3MaterialsSupplied4Precautions4IfYouHaveProblems4StorageandStability4MaterialsNeededbutNotSuppliedINTRODUCTION5Background5AboutThisAssayPRE-ASSAYPREPARATION6ReagentPreparationASSAYPROTOCOL7FlowCytometry8FluorescenceMicroscopy9PlateReaderPERFORMANCECHARACTERISTICS10RepresentativeStainingResultsRESOURCES12Troubleshooting13References14RelatedProducts15WarrantyandLimitationofRemedy16NotesGENERALINFORMATIONMaterialsSuppliedKitwillarrivepackagedasa-20°Ckit.Forbestresults,removecomponentsandstoreasstatedbelow.ItemNumberItem100TestsQuantity/SizeStorage10009908JC-1Reagent1vial/500μl-20°C10009322Cell-BasedAssayBufferTablet3tabletsRoomTemperatureIfanyoftheitemslistedabovearedamagedormissing,pleasecontactourCustomerServicedepartmentat(800)364-9897or(734)975-3999.Wecannotacceptanyreturnswithoutpriorauthorization.!WARNING:Thisproductisforlaboratoryresearchuseonly:notforadministrationtohumans.Notforhumanorveterinarydiagnosticortherapeuticuse.5INTRODUCTION4GENERALINFORMATIONPrecautionsPleasereadtheseinstructionscarefullybeforebeginningthisassay.Forresearchuseonly.Notforhumanordiagnosticuse.IfYouHaveProblemsTechnicalServiceContactInformationPhone:888-526-5351(USAandCanadaonly)or734-975-3888Fax:[phone]Email:[e-mail]:M-F8:00AMto5:30PMESTInorderforourstafftoassistyouquicklyandefficiently,pleasebereadytosupplythelotnumberofthekit(foundontheoutsideofthebox).StorageandStabilityThiskitwillperformasspecifiedifstoredasdirectedintheMaterialsSuppliedsection,onpage3,andusedbeforetheexpirationdateindicatedontheoutsideofthebox.MaterialsNeededButNotSupplied1.Adjustablepipettesandarepeatpipettor2.A6-,12-,24-,or96-wellplateforculturingcells3.Aflowcytometer,fluorescencemicroscope,orplatereaderequippedwithlaser/fluorescentfilterscapableofdetectingtheJ-aggregatesformofJC-1usinganexcitationof520-570nmandanemissionat570-610nmaswellasthemonomericformofJC-1atexcitationandemissionwavelengthsof485and535nm,respectively.4.DistilledwaterINTRODUCTIONBackgroundApotosisisacellularprocessinvolvingageneticallyprogrammedseriesofeventsleadingtothedeathofacell.Duringthisprocess,severalkeyeventsoccurinmitochondria,includingthereleaseofcaspaseactivatorssuchascytochromec,changesinelectrontransport,andlossofmitochondrialtransmembranepotential(ΔΨm).1Forthisreason,ΔΨmisanimportantparameterofmitochondrialfunctionandhasbeenusedasanindicatorofcellhealth.VariationofΔΨmhasbeenpreviouslystudiedbyevaluatingthechangesinfluorescenceintensityofcellsstainedwithcationicdyessuchasrhodamine-123(Rh123)andDiOC6.2However,dataobtainedbyusingtheseprobesmaynotbereliable.Forexample,Rh123isrelativelyinsensitivetoΔΨmchangesandDiOC6dyecannotdistinguishbetweendepolarizationoftheplasmamembraneandchangesatΔΨminseveralphysiologicalorpathologicalconditionswhenbotheventscantakeplace.3Morerecently,anewcytofluorimetric,lipophiliccationicdye,5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimi-dazolylcarbocyanineiodide(JC-1),hasbeendeveloped.JC-1hasadvantagesoverothercationicdyesinthatitcanselectivelyenterintomitochondriaandreversiblychangecolorfromgreentoredasthemembranepotentialincreases.InhealthycellswithhighmitochondrialΔΨm,JC-1spontaneouslyformscomplexesknownasJ-aggregateswithintenseredfluorescence.Ontheotherhand,inapoptoticorunhealthycellswithlowΔΨm,JC-1remainsinthemonomericform,whichshowsonlygreenfluorescence.AboutThisAssayCayman’sJC-1MitochondrialMembranePotentialAssayKitcanbeusedtostudythebehaviorofmitochondriainavarietyofconditions,includingapoptosis.ThemainadvantageofthisassayisthatthechangesinΔΨmreflectedbydifferentformsofJC-1aseithergreenorredfluorescencecanbebothqualifiedandquantifiedbyfluorescencemicroscopy,flowcytometry,orafluorescenceplatereaderwithappropriatefiltersets.7ASSAYPROTOCOL6PRE-ASSAYPREPARATIONPRE-ASSAYPREPARATIONNOTE:JC-1islightsensitive.Donotexposetodirectintenselight.ThawtheJC-1Reagentatroomtemperature.Mixwell.Toavoidrepeatedfreeze/thawingofthissolution,werecommendthatyoumakesmallaliquotsandstorethemat-20°C.ReagentPreparation1.AssayBufferPreparationDissolvethreeCell-BasedAssayBuffertablets(ItemNo.10009322)in300mlofdistilledwater.Thisbuffershouldbestableforapproximatelyoneyearatroomtemperature.2.JC-1StainingSolutionPreparationThawanaliquotoftheJC-1Reagent(ItemNo.10009908)atroomtemperature.Prepareastainingsolutionbydilutingthereagent1:10intheculturemediumyouareusingforyourcells.Mixwelltomakesuretherearenoparticlesorflakesinthesolution.NOTE:JC-1StainingSolutionisdifficulttoprepareduetoitslowsolubilityinaqueousmediumandtendencytoformparticulatesthataredifficulttoremove.MakesureJC-1Reagentiscompletelythawedandwarmedtoroomtemperaturebeforedilutingitintoculturemedium.Donotcentrifugethereagent.NOTES• JC-1 is light sensitive. All staining procedures must be performed without direct exposuretointenselight.Therefore,incubationsneedtobedoneinthedark.• For all assay protocols, on pages 7-9, it is imperative that samples be analyzed immediatelyfollowing