| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 658/675 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | NeuroBIOLOGy ReactiveOxygenSpecies |
| Related | CellSignaling CellFunctionalAnalysis BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Preparecells:
1.1 Foradherentcells:Platecellsovernightingrowthmediumat10,000to40,000cells/well/90µLfora96-wellplateor2,500to10,000cells/well/20µLfora384-wellplate.
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandsUSPendthecellpelletsinculturemediumat50,000-100,000cells/well/90µLfora96-wellpoly-Dlysineplateor10,000-25,000cells/well/20µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortoyourexperiment.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
2.PrepareROSBrite™670AssaySolution:
2.1 PrepareROSBrite™670stocksolution(500X):Add40µLofDMSO(ComponentC)intothevialofROSBrite™670(ComponentA),andmixthemwell.
Note:20µLofreconstitutedROSBrite™670stocksolutionisenoughfor1plate.Unusedportioncanbealiquotedandstoredat<-20°Cformorethanonemonthifthetubesaresealedtightlyandkeptfromlight.Avoidrepeatedfreeze-thawcycles.
2.2 PrepareROSBrite™670assaysolution:Add20µLof500XDMSOreconstitutedROSBrite™670stocksolution(fromStep2.1)into10mLofAssayBuffer(ComponentB),andmixthemwell.Thisassaysolutionisstableforatleast2hoursatroomtemperature.
3.RunROSAssay:
3.1 Treatcellswith10μLof10Xtestcompounds(96-wellplate)or5μLof5Xtestcompounds(384-wellplate)inyourdesiredbuffer(suchasPBSorHHBS).Forcontrolwells(untreatedcells),addthecorrespondingamountofcompoundbuffer.
3.2 ToinduceROS,incubatethecellplateatroomtemperatureorina5%CO2,37°Cincubatorforadesiredperiodoftime(forexample:30minutestreatmentforHelacellswith100µMtert-butylhydroperoxide(TBHP)).
3.3 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofROSBrite™670workingsolution(fromStep2.2)intothecellplate.
3.4 Incubatethecellsina5%CO2,37°Cincubatorfor30mintoonehour,andmonitorthefluorescenceincreaseatEx/Em=650/675nm(cutoff=665m)withbottomreadmode,ortakeimageswithTRITCfilterset.
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Journal:Scientificreports(2014):4554
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