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AAT Bioquest/Cell Meter™ Fluorimetric Intracellular Nitric oxide (NO) Activity Assay Kit *Orange Fluorescence Optimize
Overview | PrinterFriendlyVersion |
Ex/Em(nm) | 488/590 |
MW | N/A |
CAS# | N/A |
Solvent | N/A |
Storage | F/D/L |
Category | NeuroBIOLOGy ReactiveOxygenSpecies |
Related | CellSignaling BiochemicalAssays |
Spectrum | AdvancedSpectrumViewer |
1. Foreachsample,preparecellsin0.5mLwarmmediumorbufferofyourchoiceatadensityof5×105to1×106cells/mL.
Note:EachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforNOinduction.
2. Add1μLof500XNitrixyte™Orange(ComponentA)into0.5mLcellsUSPension.
Note:Foradherentcells,gentlyliftthecellswith0.5mMEDTAtokeepthecellsintact,andwashthecellsoncewithserum-containingmediapriortoincubationwithNitrixyte™Orange.
3. IncubatecellswithtestcompoundsandNitrixyte™Orange(fromStep2)at37ºCforadesiredperiodoftimetogenerateendogenousorexogenousNO.
Note1:Theappropriateincubationtimedependsontheindividualcelltypeandtestcompoundused.Optimizetheincubationtimeforeachexperiment.
Note2:WehaveusedRaw264.7cellsincubatedwith1XNitrixyte™Orange,20µg/mLoflipopolysaccharide(LPS)and1mML-Arginine(L-Arg)incellculturemediumat37ºCfor16hours.
4. ForaNONOatepositivecontroltreatment,add200µLofddH2OintothevialofComponentBtomake50mMstocksolution.Thestocksolutionisdilutedtomake1-2mMworkingsolutionwithAssayBuffer(ComponentC).Spindowncellsthathavepre-incubatedwithNitrixyte™Orangefor30minutes.Resuspendcellswith1mMDEANONOatepositivecontrolworkingsolution,andincubateat37ºCforanother30minutes.SeeFigure1fordetails.
5. MonitorthefluorescenceintensityattheFL2channel(Ex/Em=488/590nm)usingaflowcytometer.Gateonthecellsofinterest,excludingdebris.
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