| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 540/590 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | NeuroBIOLOGy ReactiveOxygenSpecies |
| Related | CellSignaling BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Preparecells:
1.1. Foradherentcells:Platecellsovernightingrowthmediumat30,000to80,000cells/well/90μLfora96-wellplateor8,000to20,000cells/well/20μLfora384-wellplate.
1.2. Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandsUSPendthecellpelletsinculturemediumat125,000-250,000cells/well/90µLfora96-wellpoly-Dlysineplateor30,000-60,000cells/well/20µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortoyourexperiment.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
2.Prepareworkingsolution:
2.1 Thawallthekitcomponentatroomtemperaturebeforeuse.
2.2 MakeNitrixyte™Orangeworkingsolutionforonecellplate:Add20µLofNitrixyte™Orangestocksolution(ComponentA)into10mLofAssayBufferI(ComponentB),andmixwell.Theworkingsolutionisstableforatleast2hoursatroomtemperature.
Note:20µLofNitrixyte™Orangestocksolutionisenoughforoneplate.AliquotedandstoredunusedNitrixyte™Orangestocksolutionat<-20ºC.Protectitfromlightandavoidrepeatedfreeze-thawcycles.
3.RuntheNOassay:
3.1 TostimulateendogenousNO,treatcellswith10μLof10Xtestcompounds(96-wellplate)or5μLof5Xtestcompounds(384-wellplate) incellculturemediumoryourdesiredbuffer(suchasPBSorHHBS).Forcontrolwells(untreatedcells),addthecorrespondingamountofmediumorcompoundbuffer.
Note:Itisnotnecessarytowashcellsbeforeaddingcompound.However,iftestedcompoundsareserumsensitive,growthmediumandserumfactorscanbeaspiratedawaybeforeaddingcompounds.Add90µL/well(96-wellplate)and20µL/well(384-wellplate)of1XHank’ssaltsolutionand20mMHepesbuffer(HHBS)orthebufferofyourchoiceafteraspiration.Alternatively,cellscanbegrowninserum-freemedia.
3.2 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofNitrixyte™Orangeworkingsolution(fromStep2.2)inthecellplate.Co-incubatecellswithtestcompoundandNitrixyte™Orangeworkingsolutionat37ºCfordesiredperiodoftime,protectedfromlight.
Note1:DONOTremovethetestcompounds.
Note2:ForaNONOatepositivecontroltreatment:CellswereincubatedwithNitrixyte™Orangeworkingsolutionat37ºCfor30minutes.Theworkingsolutionwasremovedandcellswerefurtherincubatedwith1mMDEA/NONOateat37ºCfor30minutestogeneratenitricoxide.SeeFigure1fordetails.
Note3:WehaveusedRaw264.7cellsincubatedwith0.5XNitrixyte™Orange,20µg/mLoflipopolysaccharide(LPS)and1mML-Arginine(L-Arg)incellculturemediumat37ºCfor16hours.SeeFigure2fordetails.
3.3 Removesolutionineachwell.AddAssayBufferII(ComponentC)100µL/wellfora96-wellplateor25µL/wellfora384-wellplate.
Note:DONOTwashcellsbeforeaddingAssayBufferII.
3.4 MonitorthefluorescenceincreaseusingmicroplatereaderatEx/Em=540/590nm(cutoff=570nm)withbottomreadmode,ortakeimagesusingfluorescencemicroscopewithaTRITCfilter.
| References&Citations |  CitationExplorer |
Fluorescentreal-timequantitativemeasurementsofintracellularperoxynitritegenerationandinhibition
Authors:ZhenLuo,QinZhao,JixiangLiu,JinfangLiao,RuoguPeng,YuntingXi,ZhenjunDiwu
Journal:AnalyticalBiochemistry(2017)
InducibleNitricOxideSynthase(iNOS)IsaNovelNegativeRegulatorofHematopoieticStem/ProgenitorCellTrafficking
Authors:MateuszAdamiak,AhmedABDelbaset-Ismail,JosephBMoore,JZhao,AhmedAbdel-Latif,MarcinWysoczynski,MariuszZRatajczak
Journal:StemCellReviewsandReports(2016):1--12
