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AAT Bioquest/Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit *Green Fluorescence*/16060/200 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 509/534 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | NeuroBIOLOGy ReactiveOxygenSpecies |
| Related | CellSignaling |
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™520workingsolution:
2.1 PrepareMitoROS™520stocksolution(500X):Add50µLofDMSO(ComponentC)intothevialofMitoROS™520(ComponentA),andmixthemwell.
Note:25μLofreconstitutedMitoROS™520stocksolutionisenoughfor1plate.Unusedportioncanbealiquotedandstoredat<-20°Cformorethanonemonthifthetubesaresealedtightlyandkeptfromlight.Avoidrepeatedfreeze-thawcycles.
2.2 PrepareMitoROS™520workingsolution:Add5μLof500XDMSOreconstitutedMitoROS™520stocksolution(fromStep2.1)into2mLofAssayBuffer(ComponentB),andmixthemwell.Thisworkingsolutionneedstobepreparedfreshlybeforeuse.Note:Theworkingsolutionisnotstable;prepareitasneededbeforeuse.
3.Runsuperoxideassay:
3.1 Treatcellswith10μLof10Xtestcompounds(96-wellplate)or5μLof5Xtestcompounds(384-wellplate)inmediumoryourdesiredbuffer(suchasPBSorHHBS).Forcontrolwells(untreatedcells),addthecorrespondingamountofcompoundbuffer.
3.2 Toinducesuperoxide,incubatethecellat37°Cforadesiredperiodoftime,protectedfromlight.
Note:WetreatedRAW264.7macrophagecellswith5µMAntimycinA(AMA)at37ºCfor2hourstoinducesuperoxide.SeeFigure1fordetails.
3.3 Add100μL/well(96-wellplate)or25μL/well(384-wellplate)ofMitoROS™520workingsolution(fromStep2.2)intothecellplate.
3.4 Incubatethecellsat37°Cfor1hour,andtakeimagesusingfluorescencemicroscopewithaFITCfilter.
| References&Citations |  PrinterFriendlyVersion |
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- GuidarelliA,FioraniM,CarloniS,CerioniL,BalduiniW,CantoniO.(2016)Thestudyofthemechanismofarsenitetoxicityinrespiration-deficientcellsrevealsthatNADPHoxidase-derivedsuperoxidepromotesthesamedownstreameventsmediatedbymitochondrialsuperoxideinrespiration-proficientcells.ToxicolApplPharmacol,307,35.
- DemaurexN,SchwarzlanderM.(2016)Mitochondrialflashes:dumpsuperoxideanddancewithprotonsnow.AntioxidRedoxSignal.
- ArcherSL.(2016)AcquiredMitochondrialAbnormalities,IncludingEpigeneticInhibitionofSuperoxideDismutase2,inPulmonaryHypertensionandCancer:TherapeuticImplications.AdvExpMedBiol,903,29.
- PorporatoPE,SonveauxP.(2015)Pavingthewayfortherapeuticpreventionoftumormetastasiswithagentstargetingmitochondrialsuperoxide.MolCellOncol,2,e968043.
- HanYH,BuffoloM,PiresKM,PeiS,SchererPE,BoudinaS.(2016)ADIpocyte-SpecificDeletionofManganeseSuperoxideDismutaseProtectsFromDiet-InducedObesityThroughIncreasedMitochondrialUncouplingandBiogenesis.Diabetes,65,2639.
- ZhongJ,XuC,Gabbay-BenzivR,LinX,YangP.(2016)Superoxidedismutase2overexpressionalleviatesmaternaldiabetes-inducedneuraltubedefects,restoresmitochondrialfunctionandsuppressescellularstressindiabeticembryopathy.FreeRadicBiolMed,96,234.
- SongSE,JoHJ,KimYW,ChoYJ,KimJR,ParkSY.(2016)Delphinidinpreventshighglucose-inducedcellproliferationandcollagensynthesisbyinhibitionofNOX-1andmitochondrialsuperoxideinmesangialcells.JPharmacolSci,130,235.
- BrandMD.(2016)Mitochondrialgenerationofsuperoxideandhydrogenperoxideasthesourceofmitochondrialredoxsignaling.FreeRadicBiolMed.
- PatelD,AlhawajR,KellyMR,AccarinoJJ,LakhkarA,GupteSA,SunD,WolinMS.(2016)PotentialroleofmitochondrialsuperoxidedecreasingferRochelataseandhemeincoronaryarterysolubleguanylatecyclasedepletionbyangiotensinII.AmJPhysiolHeartCircPhysiol,310,H1439.
