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AAT Bioquest/Cell Meter™ Intracelluar NADH/NADPH Fluorescence Imaging Kit/15290/100 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 540/590 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy CellMetabolism |
| Related | RedoxEnzymes |
| Spectrum | AdvancedSpectrumViewer |
- Preparecells:
- Foradherentcells:Platecellsovernightingrowthmediumat10,000to40,000cells/well/90μLfora96-wellplateor2,500to10,000cells/well/20μLfora384-wellplate.
- Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandsUSPendthecellpelletsinculturemediumat100,000-200,000cells/well/90µLfora96-wellpoly-Dlysineplateor25,000-50,000cells/well/20µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortoyourexperiment.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
- Prepareworkingsolution:
- Thawallthekitcomponentatroomtemperaturebeforeuse.
- MakeJZL1707NAD(P)HSensorworkingsolution:Add10µLofJZL1707NAD(P)Hsensorstocksolution(ComponentA)into2.5mLofAssayBuffer(ComponentB),andmixwell.Theworkingsolutionisstablewithin1houratroomtemperature.
Note:40µLofJZL1707NAD(P)Hsensorstocksolutionisenoughforoneplate.AliquotedandstoredunusedJZL1707NAD(P)Hsensorstocksolutionat≤-20ºC.Protectitfromlightandavoidrepeatedfreeze-thawcycles.
- RuntheNADH/NADPHassay:
- TostimulateNADP/NADPH,treatcellswith10μLof10Xtestcompounds(96-wellplate)or5μLof5Xtestcompounds(384-wellplate)inserumfreemediumoryourdesiredbuffer(suchasPBSorHHBS).Forcontrolwells(untreatedcells),addthecorrespondingamountofmediumorcompoundbuffer.
Note:JZL1707NAD(P)Hsensorisserumsensitive,thereforeit’srecommendedtokeepcellsinserum-freemediumorthebufferofyourchoice.Alternatively,cellscanbepreparedandtreatedinregularfullmedium.ChangetoserumfreemediumorbufferofyourchoicewhenincubationwithJZL1707NAD(P)Hsensor. - Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofJZL1707NAD(P)Hsensorworkingsolution(fromStep2.2)inthecellplate.Co-incubatecellswithtestcompoundandJZL1707NAD(P)Hsensorworkingsolutionat37ºCfor30-60minutes,protectedfromlight.
Note:ForaNADH/NADPHpositivecontroltreatment:HeLacellswereincubatedwith100µMNADHorNADPHfor30minutesinserum-freemedium,andco-incubatedwithJZL1707NAD(P)Hsensorworkingsolutionat37ºCforanother30minutes.SeeFigure1fordetails. - Washcellswithyourdesiredbufferonce.RemovesolutionineachwellandaddAssayBuffer(ComponentB)100µL/wellfora96-wellplateor25µL/wellfora384-wellplate.
- MonitorthefluorescenceincreaseusingmicroplatereaderatEx/Em=540/590nm(cutoff=570nm)withbottomreadmode,ortakeimagesusingfluorescencemicroscopewithaCy3®filter.
- TostimulateNADP/NADPH,treatcellswith10μLof10Xtestcompounds(96-wellplate)or5μLof5Xtestcompounds(384-wellplate)inserumfreemediumoryourdesiredbuffer(suchasPBSorHHBS).Forcontrolwells(untreatedcells),addthecorrespondingamountofmediumorcompoundbuffer.
| References&Citations |  PrinterFriendlyVersion |
1. RubinTan,JianshaLi,XiaochunPeng,LipingZhu,LeiCai,TaoWang,YuanSu,KaikobadIrani,andQinghuaHu.GAPDHiscriticalforsuperiorefficacyoffemalebonemarrow-derivedmesenchymalstemcellsonpulmonaryhypertension.CardiovascRes,Oct2013;100:19-27.
2. StephenY.Xue,ValeriaY.Hebert,DaniciaM.Hayes,CorieN.Robinson,MitziGlover,andTammyR.Dugas.NucleosideReverseTranscriptaseInhibitorsInduceaMitophagy-AssociatedEndothelialCytotoxicityThatIsReversedbyCoenzymeQ10Cotreatment.Toxicol.Sci.,Aug2013;134:323–334.
3. KateJ.Roberts,AndrewCross,OlgaVasieva,RobertJ.Moots,andStevenW.Edwards.Inhibitionofpre-Bcellcolony-enhancingfactor(PBEF/NAMPT/visfatin)decreasestheABIlityofhumanneutrophilstogeneratereactiveoxidantsbutdoesnotimpairbacterialkilling.J.Leukoc.Biol.,Sep2013;94:481-492.
4. WeijingCai,MayaRamdas,LiZhu,XueChen,GaryE.Striker,andHelenVlassara.Oraladvancedglycationendproducts(AGEs)promoteinsulinresistanceanddiabetesbydepletingtheantioxidantdefensesAGEreceptor-1andsirtuin1.PNAS,Sep2012;109:15888-15893.
5. YueQiu,ClausTittiger,ClaudeWicker-Thomas,GaëlleLeGoff,SharonYoung,EricWajnberg,ThierryFricaux,NathalieTaquet,GaryJ.Blomquist,andRenéFeyereisen.Aninsect-specificP450oxidativedecarbonylaseforcuticularhydrocarbonbiosynthesis.PNAS,Sep2012;109:14858-14863.
