AAT Bioquest/Cell Meter™ Fluorimetric Intracellular Nitric oxide (NO) Activity Assay Kit *NIR Fluorescence Optimized f,AAT Bioquest,,AAT Bioquest,aatbio,AAT Bioquest产品目录价格 AAT Bioquest试剂,,Array.Array.Array蚂蚁淘厂家直采.

产品名称：AAT Bioquest/Cell Meter™ Fluorimetric Intracellular Nitric oxide (NO) Activity Assay Kit *NIR Fluorescence Optimized f

产品编号：16359

市场价：¥100060.00

场地：美国(厂家直采)

产品分类：试剂盒>其它试剂盒>其它检测试剂盒>

联系QQ：1570468124

电话号码：4000-520-616

邮箱： info@ebiomall.com

美元价：$5003.00

品牌： AAT Bioquest

公司：AAT Bioquest

公司分类：

商品介绍

Overview

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Ex/Em(nm)	650/680
MW	N/A
CAS#	N/A
Solvent	N/A
Storage	F/D/L
Category	NeuroBIOLOGy ReactiveOxygenSpecies
Related	CellSignaling

Nitricoxide(NO)isanimportantbiologicalregulatorinvolvedinnumbersofphysiologicalandpathologicalprocesses.AlteredNOproductionisimplicatedinvariousimmunological,cardiovascular,neurodegenerativeandinflammatorydiseases.AsafreerADIcal,NOisrapidlyoxidizedandthereisrelativelylowconcentrationsofNOexistinginvivo.IthasbeenchallengingtodetectandunderstandtheroleofNOinbiologicalsystems.CellMeter™FluorimetricIntracellularNitricOxideAssayKitsprovidesensitivetoolstomonitorintracellularNOlevelinlivecells.Nitrixyte™probesaredevelopedandusedinourkitsasanexcellentreplacementforDAF-2forthedetectionandimagingoffreeNOincells.ComparedtothecommonlyusedDAF-2probe,Nitrixyte™probeshavebetterphotostABIlityandenhancedcellpermeability.ThisparticularkitusesNitrixyte™NIRthatcanreactwithNOtogeneratestrongnear-infrared(NIR)fluorescencesignal.Nitrixyte™NIRcanbereadilyloadedintolivecells,anditsfluorescencesignalcanbeconvenientlymonitoredusingthefiltersetofCy5®orAPC.Thiskitisoptimizedforfluorescenceimagingandmicroplatereaderapplications.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

Preparecells:
1. Foradherentcells:Platecellsovernightingrowthmediumat30,000to80,000cells/well/90µLfora96-wellplateor8,000to20,000cells/well/20µLfora384-wellplate.
2. Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandsUSPendthecellpelletsinculturemediumat125,000-250,000cells/well/90µLfora96-wellpoly-Dlysineplateor30,000-60,000cells/well/20µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortoyourexperiment.
  Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
Prepareworkingsolution:
1. Thawallthekitcomponentatroomtemperaturebeforeuse.
2. MakeNitrixyte™NIRworkingsolutionforonecellplate:Add20µLofNitrixyte™NIRstocksolution(ComponentA)into10mLofAssayBufferI(ComponentB),andmixwell.Theworkingsolutionisstableforatleast2hoursatroomtemperature.
  Note:20µLofNitrixyte™NIRstocksolutionisenoughforoneplate.AliquotedandstoredunusedNitrixyte™NIRstocksolutionat<-20=""°c.=""protect=""it=""from=""light=""and=""avoid=""repeated=""freeze-thaw=""cycles.="">
RuntheNOassay:
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.
2. Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofNitrixyte™NIRworkingsolution(fromStep2.2)inthecellplate.Co-incubatecellswithtestcompoundandNitrixyte™NIRworkingsolutionat37°Cfordesiredperiodoftime,protectedfromlight.
  Note1:DONOTremovethetestcompounds.
  Note2:ForaNONOatepositivecontroltreatment:CellswereincubatedwithNitrixyte™NIRworkingsolutionat37°Cfor30minutes.Theworkingsolutionwasremovedandcellswerefurtherincubatedwith1mMDEA/NONOateat37°Cfor30minutestogeneratenitricoxide.SeeFigure1fordetails.
  Note3:WehaveusedRaw264.7cellsincubatedwith0.5XNitrixyte™NIR,20µg/mLoflipopolysaccharide(LPS)and1mML-Arginine(L-Arg)incellculturemediumat37°Cfor16hours.SeeFigure2fordetails.
3. Removesolutionineachwell.AddAssayBufferII(ComponentC)100µL/wellfora96-wellplateor25µL/wellfora384-wellplate.
  Note:DONOTwashcellsbeforeaddingAssayBufferII.
4. MonitorthefluorescenceincreaseusingmicroplatereaderatEx/Em=650/680nm(cutoff=665nm)withbottomreadmode,ortakeimagesusingfluorescencemicroscopewithaCy5®filter.

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

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