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AAT Bioquest/Cell Meter™ Mitochondrial Hydroxyl Radical Detection Kit *Red Fluorescence*/16055/200 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 576/598 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | NeuroBIOLOGy ReactiveOxygenSpecies |
| Related | CellSignaling BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
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™OH580workingsolution:
2.1 PrepareMitoROS™OH580stocksolution(500X):Add50µLofDMSO(ComponentC)intothevialofMitoROS™OH580(ComponentA),andmixthemwell.
Note:25μLofreconstitutedMitoROS™OH580stocksolutionisenoughfor1plate.Unusedportioncanbealiquotedandstoredat≤-20ºCformorethanonemonthifthetubesaresealedtightlyandkeptfromlight.Avoidrepeatedfreeze-thawcycles.
2.2 PrepareMitoROS™OH580workingsolution:Add25μLof500XDMSOreconstitutedMitoROS™OH580stocksolution(fromStep2.1)into10mLofAssayBuffer(ComponentB),andmixthemwell.Thisworkingsolutionisstableforatleast2hoursatroomtemperature.
3.Runhydroxylradicalassay:
3.1 Removemedium,andadd100μL/well(96-wellplate)or25μL/well(384-wellplate)ofMitoROS™OH580workingsolution(fromStep2.2)intothecellplate.Incubatecellsat37ºCfor1hour.
3.2 Toinducehydroxylradical,treatcellswithtextcompoundsinyourdesiredbuffer(suchasPBSorHHBS)at37ºCforadesiredperiodoftime,protectedfromlight.
Note1:WetreatedHeLacellswithFentonreaction(10µMCuCl2and100µMH2O2)at37ºCfor1hourtoinduceexogenoushydroxylradical.SeeFigure1fordetails.
Note2:WetreatedRAW264.7cellswithPMA(phorbol12-myristate13-acetate)ingrowthmediumat37ºCfor4hourstostimulateendogenoushydroxylradical.SeeFigure2fordetails.
3.3 Washcells2-3timeswithHHBSorDPBS,andadd100μLAssayBuffer(ComponentB)toeachwell.
3.4 MonitorthefluorescencesignalincellsusingfluorescencemicroscopewithaTRITCfilterset,ormeasurefluorescenceincreaseusingfluorescencemicroplatereaderatEx/Em=540/590nm(cutoff=570nm)withbottomreadmode.
| References&Citations |  PrinterFriendlyVersion |
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- FrancesDE,RoncoMT,MontiJA,IngaramoPI,PisaniGB,ParodyJP,PellegrinoJM,SanzPM,CarrilloMC,CarnovaleCE.(2010)Hyperglycemiainducesapoptosisinratliverthroughtheincreaseofhydroxylradical:newinsightsintotheinsulineffect.JEndocrinol,205,187.
- ZhangX,MonroeME,ChenB,ChinMH,HeibeckTH,SchepmoesAA,YangF,PetritisBO,CampDG,2nd,PoundsJG,JacobsJM,SmithDJ,BigelowDJ,SmithRD,QianWJ.(2010)Endogenous3,4-dihydroxyphenylalanineanddopaquinonemodificationsonproteintyrosine:linkstomitochondriallyderivedoxidativestressviahydroxylradical.MolCellProteomics,9,1199.
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