| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 520/605 |
| 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/100µLfora96-wellplateor2,500to10,000cells/well/25µLfora384-wellplate.
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandsUSPendthecellpelletsinculturemediumat50,000-100,000cells/well/100µLfora96-wellpoly-Dlysineplateor10,000-25,000cells/well/25µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortoyourexperiment.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
2.PrepareAmplite™ROSRedstainsolution:
2.1 PrepareAmplite™ROSRedstocksolution(500X):Add40µLofDMSO(ComponentC)intothevialofAmplite™ROSRed(ComponentA),andmixthemwell.
Note:20µLofreconstitutedAmplite™ROSRedstocksolutionisenoughfor1plate.Unusedportioncanbealiquotedandstoredat<-20°Cformorethanonemonthifthetubesaresealedtightlyandkeptfromlight.Avoidrepeatedfreeze-thawcycles.
2.2 PrepareAmplite™ROSRedworkingsolution:Add20µLof500XDMSOreconstitutedAmplite™ROSRedstocksolution(fromStep2.1)into10mLofAssayBuffer(ComponentB),andmixthemwell.Thisworkingsolutionisstableforatleast2hoursatroomtemperature.
3.Stainthecells:
3.1 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofAmplite™ROSRedworkingsolution(fromStep2.2)intothecellplate.
3.2 Incubatethecellsina5%CO2,37°Cincubatorforonehour.
4.RunROSAssay:
4.1 Treatcellswith20μLof11Xtestcompounds(96-wellplate)or10μLof6Xtestcompounds(384-wellplate)inyourdesiredbuffer(suchasPBSorHHBS).Forcontrolwells(untreatedcells),addthecorrespondingamountofcompoundbuffer.
4.2 ToinduceROS,incubatethecellplateatroomtemperatureorina5%CO2,37°Cincubatorforatleast15minutesoradesiredperiodoftime(30minutesforHelacellstreatedwith1mMH2O2).
4.3 MonitorthefluorescenceincreaseatEx/Em=520/605nmwithbottomreadmode.
| References&Citations |  CitationExplorer |
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Journal:Scientificreports(2014):4554
Highglucose-inducedmitochondrialrespirationandreactiveoxygenspeciesinmousecerebralpericytesisreversedbypharmacologicalinhibitionofmitochondrialcarbonicanhydrases:implicationsforcerebralmicrovasculardiseaseindiabetes
Authors:GulNShah,YoichiMorofuji,WilliamABanks,TulinOPrice
Journal:Biochemicalandbiophysicalresearchcommunications(2013):354--358
