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AAT Bioquest/Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit*Optimized for Flow Cytometry*/22970/
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 540/590 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | NeuroBIOLOGy ReactiveOxygenSpecies |
| Related | CellSignaling CellFunctionalAnalysis BiochemicalAssays |
1.Preparecells:
Foreachsample,preparecellsin0.5mLgrowthmediumorbufferofyourchoiceatadensityof5×105to1×106cells/mL.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforsuperoxideinduction.
2.PrepareMitoROS™580stocksolution:
Add100µLofDMSO(ComponentC)intothevialofMitoROS™580(ComponentA),andmixthemwell.
Note:1μLofreconstitutedMitoROS™580stocksolutionisfor0.5mLcells.Unusedportioncanbealiquotedandstoredat<-20°Cformorethanonemonthifthetubesaresealedtightlyandkeptfromlight.Avoidrepeatedfreeze-thawcycles.
3.Runsuperoxideassay:
3.1Teatcellswith25µLof 20XtestcompoundsinAssayBuffer(ComponentB)oryourdesiredbuffer(suchasPBS)toinducesuperoxide.Forcontrolcells(untreatedcells),addthecorrespondingamountofcompoundbuffer.
3.2Incubatethecellsat37°Cforatleast30minutesoradesiredperiodoftime,protectedfromlight.
Note:Jurkatcellsweretreatedwith50µMAntimycinA(AMA)at37ºCfor30minutestoinducesuperoxide.SeeFigure1fordetails. Pyocyanin(50µM)orH2O2(1mM)canalsobeusedtoinducesuperoxide.
3.3Add1μLof500XMitoROS™580(ComponentA)into0.5mLcellsUSPension,andincubateat37ºCfor1hour.
Note1:Foradherentcells,gentlyliftthecellswith0.5mMEDTAtokeepthecellsintact,andwashthecellsoncewithserum-containingmediapriortoincubationwithMitoROS™580.
Note2:Theappropriateincubationtimedependsontheindividualcelltypeandtestcompoundused.Optimizetheincubationtimeforeachexperiment.
3.4Incubatethecellsat37°Cfor30minto1hour.MonitorthefluorescenceintensityattheFL2channel
(Ex/Em=488/590nm)usingaflowcytometer.Gateonthecellsofinterest,excludingdebris.
| References&Citations |  PrinterFriendlyVersion |
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