Overview | ![]() PrinterFriendlyVersion |
Ex/Em(nm) | 502/535 |
MW | N/A |
CAS# | N/A |
Solvent | N/A |
Storage | F/D/L |
Category | NeuroBIOLOGy ReactiveOxygenSpecies |
Related | CellSignaling BiochemicalAssays |
Spectrum | AdvancedSpectrumViewer |
1.Preparecells:
Foreachsample,preparecellsin0.5mLgrowthmediumorbufferofyourchoiceatadensityof5×105to1×106cells/mL.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforperoxynitriteinduction.
2.Prepare400XofDAX-J2™PONGreenstocksolution:
Add25µLofDMSO(ComponentB)intothevialofDAX-J2™PONGreen(ComponentA),andmixthemwell.
Note:1μLofreconstitutedDAX-J2™PONGreenstocksolutionisfor0.4mLcells.Unusedportioncanbealiquotedandstoredat≤-20°Cformorethanonemonthifthetubesaresealedtightlyandkeptfromlight.Avoidrepeatedfreeze-thawcycles.
3.Runtheperoxynitriteassay:
3.1 Co-incubatecellswithDAX-J2™PONGreenwithtestcompoundsinfullmediumorinyourdesiredbufferat37ºCfordesiredperiodoftime,protectedfromlight.Forcontrolwells(untreatedcells),addthecorrespondingamountofcompoundbuffer.
Note1:It’srecommendedtostainthecellsinfullmedium.However,iftestedcompoundsareserumsensitive,growthmediumandserumfactorscanbeaspiratedawaybeforestaining.ResUSPendcellsin1XHank’ssaltsolutionand20mMHepesbuffer(HHBS)orthebufferofyourchoiceafteraspiration.Alternatively,cellscanbestainedinserum-freemedia.
Note2:Weco-incubatedRAW264.7macrophagecellswith50-200μMSIN-1andDAX-J2™PONGreeninfullmediumat37ºCfor1hourtoinduceperoxynitrite.SeeFigure1(A)fordetails.
3.2 Alternatively,staincellswithDAX-J2™PONGreenat37ºCfor1hour,protectedfromlight(asinStep3.1).Removetheworkingsolution,thentreatcellswithtestcompoundsinfullmediumorinyourdesiredbufferat37ºCfordesiredperiodoftime.SeeFigure1(B)fordetails.
3.3 MonitorthefluorescenceintensityatFITCchannel(Ex/Em=490/530nm)usingaflowcytometer.Gateonthecellsofinterest,excludingdebris.
References&Citations | ![]() CitationExplorer |
Fluorescentreal-timequantitativemeasurementsofintracellularperoxynitritegenerationandinhibition
Authors:ZhenLuo,QinZhao,JixiangLiu,JinfangLiao,RuoguPeng,YuntingXi,ZhenjunDiwu
Journal:Analyticalbiochemistry(2017):44--48
GenesilencingofendothelialvonWillebrandFactorattenuatesangiotensinII-inducedendothelin-1expressioninporcineaorticendothelialcells
Authors:AnarDushpanova,SilviaAgostini,EnricaCiofini,ManuelaCABIati,ValentinaCasieri,MarcoMatteucci,SilviaDelRy,AldoClerico,SergioBerti,VincenzoLionetti
Journal:ScientificReports(2016)
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