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AAT Bioquest/Cell Meter™ Fluorimetric Intracellular Peroxynitrite Assay Kit *Green Fluorescence*/16315/100 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 502/535 |
| MW | N/A |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | NeuroBIOLOGy ReactiveOxygenSpecies |
| Related | CellSignaling BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Preparecells:
1.1. Foradherentcells:Platecellsovernightingrowthmediumat20,000to80,000cells/well/90μLfora96-wellplateor5,000to20,000cells/well/22.5μLfora384-wellplate.
1.2. Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandsUSPendthecellpelletsinculturemediumat80,000-200,000cells/well/90µLfora96-wellpoly-Dlysineplateor20,000-50,000cells/well/22.5µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortoyourexperiment.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
2.Prepareworkingsolution:
2.1 PrepareDAX-J2™PONGreenstocksolution(500X):Add20µLofDMSO(ComponentC)intothevialofDAX-J2™PONGreen(ComponentA),andmixthemwell.
Note:20μLofreconstitutedDAX-J2™PONGreenstocksolutionisenoughfor1plate.Unusedportioncanbealiquotedandstoredat≤-20°Cformorethanonemonthifthetubesaresealedtightlyandkeptfromlight.Avoidrepeatedfreeze-thawcycles.
2.2 PrepareDAX-J2™PONGreenworkingsolution(10X):Add10μLof500XDMSOreconstitutedDAX-J2™PeroxynitriteSensorstocksolution(fromStep2.1)into500μLofAssayBuffer(ComponentB),andmixthemwell.
Note:Theworkingsolutionisnotstable;prepareitasneededbeforeuse.
3.Runtheperoxynitriteassay:
3.1 Add10µL/well(96-wellplate),or2.5µL/well(384-wellplate)ofDAX-J2™PONGreenworkingsolution(fromStep2.2)in90µL(96-wellplate)or22.5µL(384-wellplate)cellcultureperwellinthecellplate(fromStep1).
Note:Itisnotnecessarytowashcellsbeforestaining.It’srecommendedtostainthecellsinfullmedium.
3.2 Co-incubatecellswithDAX-J2™PONGreenwithtestcompoundsinfullmediumorinyourdesiredbufferat37ºCfordesiredperiodoftime,protectedfromlight.Forcontrolwells(untreatedcells),addthecorrespondingamountofcompoundbuffer.
Note1:It’srecommendedtostainthecellsinfullmedium.However,iftestedcompoundsareserumsensitive,growthmediumandserumfactorscanbeaspiratedawaybeforestaining.Add90µL/well(96-wellplate)and22.5µL/well(384-wellplate)of1XHank’ssaltsolutionand20mMHepesbuffer(HHBS)orthebufferofyourchoiceafteraspiration.Alternatively,cellscanbestainedinserum-freemedia.
Note2:Weco-incubatedRAW264.7macrophagecellswith50-200μMSIN-1andDAX-J2™PONGreeninfullmediumat37ºCfor1hourtoinduceperoxynitrite.SeeFigure1fordetails.
3.3 Alternatively,staincellswithDAX-J2™PONGreenat37ºCfor1hour,protectedfromlight(asinStep3.1).Removetheworkingsolution,thentreatcellswithtestcompoundsinfullmediumorinyourdesiredbufferat37ºCfordesiredperiodoftime.
3.4 MonitorthefluorescenceincreaseusingmicroplatereaderatEx/Em=490/530nm(cutoff=515nm)withbottomreadmode,ortakeimagesusingfluorescencemicroscopewithaFITCfilter.
| 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)
