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AAT Bioquest/Cell Meter™ Mitochondrion Membrane Potential Assay Kit *Orange Fluorescence Optimized for Microplate Read
| 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 |
1.Preparecells:
1.1 Foradherentcells:Platecellsovernightingrowthmediumat20,000to80,000cells/well/100µLfora96-wellplateor5,000to20,000cells/well/25µLfora384-wellplate.
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletsinculturemediumat100,000-200,000cells/well/90μLfora96-wellpoly-Dlysineplateor25,000-50,000cells/well/20μLfora384-wellpoly-Dlysineplate.Centrifugetheplatesat800rpmfor2minuteswithbrakeoffpriortotheexperiments.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforapoptosisinduction
2.PrepareMitoTell™Orangedye-loadingsolution:
2.1 Thawallthekitcomponentsatroomtemperaturebeforeuse.
2.2 Add50µLof200XMitoTell™Orange(ComponentA)into10mLofAssayBufferA(ComponentB),andmixthemwell.
Note:Aliquotandstoretheunused200XMitoTell™Orange(ComponentA)at-20oC.Avoidrepeatedfreeze/thawcycles.
3.RunMitoTell™OrangeAssay:
3.1 Treatcellswithtestcompoundsforadesiredperiodoftimetoinduceapoptosis,andsetupparallelcontrolexperiments.
ForNegativeControl:Treatcellswithvehicleonly.
ForPositiveControl:TreatcellswithFCCPorCCCPat5-50µMina5%CO2,37oCincubatorfor15to30minutes.
Note:CCCPorFCCPcanbeaddedsimultaneouslywithMitoTell™Orange.Togetthebestresult,titrationoftheCCCPorFCCPmayberequiredforeachindividualcellline.
3.2 Removethecellmedium.
Note:ItisimportanttoremovethecellmediumbeforeaddingMitoTell™Orangedye-loadingsolution.
3.3 Add100µL/well/96-wellplateor25µL/well/384-wellplateofMitoTell™Orangedye-loadingsolution(fromStep2.2)intothecellplate(fromStep3.2).
3.4 Incubatethedye-loadingplateina5%CO2,37oCincubatorfor15-30minutes,protectedfromlight.
Note:Theappropriateincubationtimedependsontheindividualcelltypeandcellconcentrationused.Optimizetheincubationtimeforeachexperiment.
3.5 Add50µL/well/96-wellplateor12.5µL/well/384-wellplateofAssayBufferB(ComponentC)intothedye-loadedcellplate(fromStep3.4).
Note1:DONOTwashthecellsafterloading.
Note2:Fornon-adherentcells,itisrecommendedtocentrifugecellplatesat800rpmfor2minuteswithbrakeoffafteraddingAssayBufferB(ComponentC).
3.6 MonitorthefluorescenceintensityatEx/Em=540/590nm(bottomread)10to30minutesafterStep3.5eitherusingtheendpointmodeorusingthekineticmode.
| 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.
