| 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.Prepare500XDAX-J2™PONGreen99stocksolution:
Add20µLofDMSO(ComponentC)intothevialofDAX-J2™PONGreen99(ComponentA)tomake500Xstocksolution.
Note:20μLofreconstitutedDAX-J2™PONGreen99stocksolutionisenoughfor1plate.Unusedportioncanbealiquotedandstoredat≤-20°Cformorethanonemonthifthetubesaresealedtightlyandkeptfromlight.Avoidrepeatedfreeze-thawcycles.
2.PrepareONOO¯assaymixture:
Add20µLof500XDAX-J2™PONGreen99stocksolution(fromStep1)into10mLofAssayBuffer(ComponentB),andmixthemwell.
Note:Thisassaymixtureisenoughforone96-wellplate.Itisstableat4ºCfor2hourswhenprotectedfromlight.
3.PrepareONOO¯stocksolution(notprovided):
Peroxynitritestocksolutionwassynthesizedaccordingtoliteraturereport.Briefly,amixtureofsodiumnitrite(0.6M)andhydrogenperoxide(0.7M)wasacidifiedwithhydrochloricacid(0.6M),andsodiumhydroxide(1.5M)wasaddedwithin1-2secondstomakethesolutionalkaline.Theexcesshydrogenperoxidewasremovedbypassingthesolutionthroughashortcolumnofmanganesedioxide.Theextinctioncoefficientofperoxynitritesolutionin0.1MNaOHis1670M-1cm-1at302nm.TheONOO¯stocksolutionisnotstable;youmightstoreitat≤-80°Cfor2-4weeks,wehighlyrecommendmakeitfreshtouse.
4.PrepareserialdilutionsofONOO¯standard(0to20µM):
4.1 DiluteONOO¯stocksolution(fromStep3)inAssaybuffer(ComponentB)tohave20µMONOO¯standardsolution,andthenperform1:2serialdilutionstogetapproximately20,10,5,2.5,1.25,0.625,0.313and0µM.
4.2 AddONOO¯standardsandONOO¯containingtestsamplesintoa96-wellsolidblackmicroplateasdescribedinTables1and2.
Table1.LayoutofONOO¯standardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
|
|
|
|
|
|
O1 | O1 | …. | …. | …. | …. |
|
|
|
|
|
|
O2 | O2 |
|
|
|
|
|
|
|
|
|
|
O3 | O3 |
|
|
|
|
|
|
|
|
|
|
O4 | O4 |
|
|
|
|
|
|
|
|
|
|
O5 | O5 |
|
|
|
|
|
|
|
|
|
|
O6 | O6 |
|
|
|
|
|
|
|
|
|
|
O7 | O7 |
|
|
|
|
|
|
|
|
|
|
*Note:O=ONOO¯Standards;BL=BlankControl;TS=TestSamples
Table2.Reagentcompositionforeachwell
ONOO¯Standard | BlankControl | TestSample |
Serialdilutions*(50μL) | TE:50μL | 50μL |
*Note:AddtheseriallydilutionsofONOO¯standardsfrom0.313to20µmintowellsfromO1toO7induplicate.
5.RunONOO¯assay:
5.1 Add50μLofassaymixture(fromStep2)toeachwelloftheONOO¯standard,blankcontrol,andtestsamples(seeStep4.2)tomakethetotalassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLsampleand25μLofassaymixtureperwell.
5.2 Incubatethereactionatroomtemperaturefor5to10minutes,protectedfromlight.
5.3 MonitorthefluorescenceincreaseatEx/Em=490/530nm(cutoffat515nm)withafluorescenceplatereader.
| 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)
