| 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
AAT Bioquest常用产品报价单
| 产品名 | 货号 | 公司分类 | 商城分类 | 美金价 |
| AAT Bioquest/Fluorescein, disodium salt *CAS 518-47-8*/2/100 mg | 2 | iFluor Rapid Tests | 酸碱缓冲液 | 1088.00 | AAT Bioquest/14-3-3 ζ (Ab-58) Antibody/8B0001/50 ug | 8B0001 | Signaling Intermediate Ab | 重组抗体 | 2828.00 | AAT Bioquest/Opioid Receptor (Phospho-Ser375) Antibody/8A0022/50 ug | 8A0022 | Phospho-Specific Ab | 受体 | 2828.00 | AAT Bioquest/iFluor™ 350 goat anti-mouse IgG (H+L)/16440/200 ug | 16440 | Fluorescent Anti-IgGs | 荧光染料 | 1088.00 | AAT Bioquest/trFluor™ Eu goat anti-mouse IgG (H+L)/16518/100 ug | 16518 | Fluorescent Anti-IgGs | 抗小鼠 | 4278.00 | AAT Bioquest/RPE-streptavidin conjugate/16900/100 ug | 16900 | Streptavidin Conjugates | 其他生物染料 | 1378.00 | AAT Bioquest/GCNT3 Antibody/8C14708/50 ug | 8C14708 | Signaling Intermediate Ab | 功能性抗体 | 2828.00 | AAT Bioquest/Amplite™ Luciferase Reporter Gene Assay Kit *Bright Glow*/12518/1 plate | 12518 | Reporter Gene Enzymes | 其它检测试剂盒 | 2103.00 | AAT Bioquest/ReadiLink™ Rapid mFluor™ Violet 450 Antibody Labeling Kit *Microscale Optimized for Labeling 50 &mi | 1100 | mFluor™ Dyes and Kits | 标记试剂盒 | 2103.00 | AAT Bioquest/Amplite™ Colorimetric Urea Quantitation Kit *Blue Color*/10058/200 Tests | 10058 | Diagnostic Molecules | 定量试剂盒 | 2828.00 | AAT Bioquest/Screen Quest™ CHO-Gqi Chimera Cell line/38101/Each | 38101 | cAMP GPCR Assays | 常规细胞株 | 0.00 |
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