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AAT Bioquest/Cell Meter™ Mitochondrion Membrane Potential Assay Kit *Orange Fluorescence Optimized for Flow Cytometry*
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 546/575 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellAnalysis CellCytotoxicity |
| Related | ApoptosisandCytotoxicity CellApoptosis BiochemicalAssays |
AssayProtocolforFlowCytometer
Note:Thawallthekitcomponentsatroomtemperaturebeforeuse.
1.Foreachsample,preparecellsin1mLofwarmmediumorbufferofyourchoiceatthedensityof5×105to1×106cells/mL.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforapoptosisinduction.
2.Treatcellswithtestcompoundsforadesiredperiodoftimetoinduceapoptosis,andsetupparallelcontrolexperiments.
ForNegativeControl:Treatcellswithvehicleonly.
ForPositiveControl:TreatcellswithFCCPorCCCPat5-50µMina37oC,5%CO2incubatorfor15to30minutes.
Note:CCCPorFCCPcanbeaddedsimultaneouslywithMitoTell™Orange(SeeStep3).Togetthebestresult,titrationoftheCCCPorFCCPmayberequiredforeachindividualcellline.
3.Add2μLof500XMitoTell™Orange(ComponentA)intothetreatedcells(fromStep2),andincubatethecellsina37°C,5%CO2incubatorfor15to30minutes.
Note:Foradherentcells,gentlyliftthecellswith0.5mMEDTAtokeepthecellsintactandwashthecellsoncewithserum-containingmediapriortotheincubationwithMitoTell™Orangedye-loadingsolution.
4.Centrifugethecellsat1000rpmfor4minutes,andthenre-sUSPendcellsin1mLofAssayBuffer(ComponentB)orbufferofyourchoice.
5.MonitorthefluorescenceintensitybyusingaflowcytometerintheFL2channel(Ex/Em=540/590nm).Gateonthecellsofinterest,excludingdebris.
| References&Citations |  PrinterFriendlyVersion |
1. PerevoshchikovaIV,SorochkinaAI,ZorovDB,AntonenkoYN.(2009)SafranineOasafluorescentprobeformitochondrialmembranepotentialstudiedonthesingleparticlelevelandinsuspension.Biochemistry(Mosc),74,663.
2. ChalmersS,McCarronJG.(2008)ThemitochondrialmembranepotentialandCa2+oscillationsinsmoothmuscle.JCellSci,121,75.
3. DistelmaierF,KoopmanWJ,TestaER,deJongAS,SwartsHG,MayatepekE,SmeitinkJA,WillemsPH.(2008)Lifecellquantificationofmitochondrialmembranepotentialatthesingleorganellelevel.CytometryA,73,129.
4. GuthrieHD,WelchGR.(2008)Determinationofhighmitochondrialmembranepotentialinspermatozoaloadedwiththemitochondrialprobe5,5",6,6"-tetrachloro-1,1",3,3"-tetraethylbenzimidazolyl-carbocyanineiodide(JC-1)byusingfluorescence-activatedflowcytometry.MethodsMolBiol,477,89.
5. KoopmanWJ,DistelmaierF,EsselingJJ,SmeitinkJA,WillemsPH.(2008)Computer-assistedlivecellanalysisofmitochondrialmembranepotential,morphologyandcalciumhandling.Methods,46,304.
6. LabiosM,MartinezM,GabrielF,GuiralV,Ruiz-AjaS,BeltranB,MunozA.(2008)EffectsofeprosartanonmitochondrialmembranepotentialandH2O2levelsinleucocytesinhypertension.JHumHypertens,22,493.
7. RamadassR,Bereiter-HahnJ.(2008)HowDASPMIrevealsmitochondrialmembranepotential:fluorescencedecaykineticsandsteady-stateanisotropyinlivingcells.BiophysJ,95,4068.
8. VerburgJ,HollenbeckPJ.(2008)Mitochondrialmembranepotentialinaxonsincreaseswithlocalnervegrowthfactororsemaphorinsignaling.JNeurosci,28,8306.
9. XiaXY,WuYM,HouBS,YangB,PanLJ,ShiYC,JinBF,ShaoY,CuiYX,HuangYF.(2008)[EvaluationofspermmitochondrialmembranepotentialbyJC-1fluorescentstainingandflowcytometry].ZhonghuaNanKeXue,14,135.
10. AndersenAZ,PoulsenAK,BrasenJC,OlsenLF.(2007)On-linemeasurementsofoscillatingmitochondrialmembranepotentialinglucose-fermentingSaccharomycescerevisiae.Yeast,24,731.
