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AAT Bioquest/Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit*Optimized for Microplate Reader*/229
| 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:
1.1 Foradherentcells:Platecellsovernightingrowthmediumat10,000to40,000cells/well/90μLfora96-wellplateor2,500to10,000cells/well/20μLfora384-wellplate.
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandsUSPendthecellpelletsinculturemediumat100,000-200,000cells/well/90µLfora96-wellpoly-Dlysineplateor25,000-50,000cells/well/20µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortoyourexperiment.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
2.PrepareMitoROS™580workingsolution:
2.1 PrepareMitoROS™580stocksolution(500X):Add50µLofDMSO(ComponentC)intothevialofMitoROS™580(ComponentA),andmixthemwell.
Note:25μLofreconstitutedMitoROS™580stocksolutionisenoughfor1plate.Unusedportioncanbealiquotedandstoredat<-20°Cformorethanonemonthifthetubesaresealedtightlyandkeptfromlight.Avoidrepeatedfreeze-thawcycles.
2.2 PrepareMitoROS™580workingsolution:Add25μLof500XDMSOreconstitutedMitoROS™580stocksolution(fromStep2.1)into10mLofAssayBuffer(ComponentB),andmixthemwell.Thisworkingsolutionisstableforatleast2hoursatroomtemperature.
3.Runsuperoxideassay:
3.1 Treatcellswith10μLof10Xtestcompounds(96-wellplate)or5μLof5Xtestcompounds(384-wellplate)inyourdesiredbuffer(suchasPBSorHHBS).Forcontrolwells(untreatedcells),addthecorrespondingamountofcompoundbuffer.
3.2 Toinducesuperoxide,incubatethecellplateat37°Cforadesiredperiodoftime,protectedfromlight.
Note:WetreatedHeLacellswith50µMAntimycinA(AMA)at37ºCfor30minutestoinducesuperoxide.SeeFigure1fordetails.
3.3 Add100μL/well(96-wellplate)or25μL/well(384-wellplate)ofMitoROS™580workingsolution(fromStep2.2)intothecellplate.
3.4 Incubatethecellsat37°Cfor30minto1hour,andmonitorthefluorescenceincreaseatEx/Em=540/590nm(cutoff=570m)withbottomreadmode,ortakeimagesusingfluorescencemicroscopewithaTRITCfilter.
| References&Citations |  PrinterFriendlyVersion |
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