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主营:研究并生产荧光和发光探针,信号转导研究的试剂
℡ 4000-520-616
℡ 4000-520-616
AAT Bioquest/Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit*Optimized for Microplate Reader*/229
产品编号:22971
市  场 价:¥85560.00
场      地:美国(厂家直采)
联系QQ:1570468124
电话号码:4000-520-616
邮      箱: info@ebiomall.com
美  元  价:$4278.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit*Optimized for Microplate Reader*/229
商品介绍
Overview
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Ex/Em(nm)540/590
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryNeuroBIOLOGy
ReactiveOxygenSpecies
RelatedCellSignaling
CellFunctionalAnalysis
BiochemicalAssays
Mitochondriaaremajorproducersofcellularsuperoxide.Theproductionoflowtomoderatelevelsofsuperoxideiscriticalfortheproperregulationofmanyessentialcellularprocessesincludinggeneexpression,signaltransduction,andmuscleadaptationtoenduranceexercisetraining.Uncontrolledmitochondrialsuperoxideproductioncantriggercellularoxidativedamagethatcontributestothepathogenesisofawidevarietyofdisordersincludingcancer,cardiovasculardiseases,neurodegenerativediseasesandaging.Thedetectionofintracellularmitochondrialsuperoxideisofcentralimportancetounderstandingpropercellularredoxregulationandtheimpactofitsdysregulationonvariouspathologies.CellMeter™FluorimetricMitochondrialSuperoxideActivityAssayKitusesouruniqueSuperoxideIndicatortoquantifysuperoxidelevelinlivecells.MitoROS™580islive-cellpermeantandcanrapidlyandselectivelytargetsuperoxideinmitochondria.Itgeneratesredfluorescencewhenitreactswithsuperoxide,andcanbeeasilyreadatEx/Em=540/590nm.TheCellMeter™FluorimetricIntracellularSuperoxideDetectionKitprovidesasensitive,one-stepfluorimetricassaytodetectmitochondrialsuperoxideinlivecellswithonehourincubation.Thiskitcanbeusedforfluorescencemicroplatereadersandfluorescencemicroscopyapplications.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.Preparecells:

1.1   Foradherentcells:Platecellsovernightingrowthmediumat10,000to40,000cells/well/90μLfora96-wellplateor2,500to10,000cells/well/20μLfora384-wellplate.

1.2   Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandsUSPendthecellpelletsinculturemediumat100,000-200,000cells/well/90µLfora96-wellpoly-Dlysineplateor25,000-50,000cells/well/20µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortoyourexperiment.

Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.

 

2.PrepareMitoROS™580workingsolution:

2.1   PrepareMitoROS™580stocksolution(500X):Add50µLofDMSO(ComponentC)intothevialofMitoROS™580(ComponentA),andmixthemwell. 

Note:25μLofreconstitutedMitoROS™580stocksolutionisenoughfor1plate.Unusedportioncanbealiquotedandstoredat<-20°Cformorethanonemonthifthetubesaresealedtightlyandkeptfromlight.Avoidrepeatedfreeze-thawcycles.

2.2   PrepareMitoROS™580workingsolution:Add25μLof500XDMSOreconstitutedMitoROS™580stocksolution(fromStep2.1)into10mLofAssayBuffer(ComponentB),andmixthemwell.Thisworkingsolutionisstableforatleast2hoursatroomtemperature.

 

3.Runsuperoxideassay:

3.1   Treatcellswith10μLof10Xtestcompounds(96-wellplate)or5μLof5Xtestcompounds(384-wellplate)inyourdesiredbuffer(suchasPBSorHHBS).Forcontrolwells(untreatedcells),addthecorrespondingamountofcompoundbuffer.

3.2   Toinducesuperoxide,incubatethecellplateat37°Cforadesiredperiodoftime,protectedfromlight.

Note:WetreatedHeLacellswith50µMAntimycinA(AMA)at37ºCfor30minutestoinducesuperoxide.SeeFigure1fordetails.

3.3   Add100μL/well(96-wellplate)or25μL/well(384-wellplate)ofMitoROS™580workingsolution(fromStep2.2)intothecellplate.

3.4   Incubatethecellsat37°Cfor30minto1hour,andmonitorthefluorescenceincreaseatEx/Em=540/590nm(cutoff=570m)withbottomreadmode,ortakeimagesusingfluorescencemicroscopewithaTRITCfilter.

References&Citations
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1.   KitamuraK,MaruyamaK,HamanoS,KishiT,KawakamiT,TakahashiY,OnoderaS.(2014)Effectofhypochloriteoxidationoncholinesterase-inhibitionassayofacetonitrileextractsfromfruitsandvegetablesformonitoringtracesoforganophosphatepesticides.JToxicolSci,39,71.

2.   MartinDE,DeAlmeidaJF,HenryMA,KhaingZZ,SchmidtCE,TeixeiraFB,DiogenesA.(2014)Concentration-dependenteffectofsodiumhypochloriteonstemcellsofapicalpapillasurvivalanddifferentiation.JEndod,40,51.

3.   YinB,DengJ,PengX,LongQ,ZhaoJ,LuQ,ChenQ,LiH,TangH,ZhangY,YaoS.(2013)Greensynthesisofcarbondotswithdown-andup-conversionfluorescentpropertiesforsensitivedetectionofhypochloritewithadual-readoutassay.Analyst,138,6551.

4.   TakimotoK,TaharaguchiM,SakaiK,TakagiH,TohyaY,YamadaYK.(2013)Effectofhypochlorite-baseddisinfectantsoninactivationofmurinenorovirusandattempttoeliminateorpreventinfectioninmicebyadditiontodrinkingwater.ExpAnim,62,237.

5.   SetlowB,YuJ,LiYQ,SetlowP.(2013)AnalysisofthegerminationkineticsofindividualBacillussubtilissporestreatedwithhydrogenperoxideorsodiumhypochlorite.LettApplMicrobiol,57,259.

6.   ThornRM,RobinsonGM,ReynoldsDM.(2013)Comparativeantimicrobialactivitiesofaerosolizedsodiumhypochlorite,chlorinedioxide,andelectRochemicallyactivatedsolutionsevaluatedusinganovelstandardizedassay.AntimicrobAgentsChemother,57,2216.

7.   Perez-CruzF,CortesC,AtalaE,BohleP,ValenzuelaF,Olea-AzarC,SpeiskyH,AspeeA,LissiE,Lopez-AlarconC,BridiR.(2013)Useofpyrogallolredandpyranineasprobestoevaluateantioxidantcapacitiestowardshypochlorite.Molecules,18,1638.

8.   ZhangJ,YangX.(2013)Asimpleyeteffectivechromogenicreagentfortherapidestimationofbromateandhypochloriteindrinkingwater.Analyst,138,434.

9.   CollaoB,MoralesEH,GilF,PolancoR,CalderonIL,SaavedraCP.(2012)DifferentialexpressionofthetranscriptionfactorsMarA,Rob,andSoxSofSalmonellaTyphimuriuminresponsetosodiumhypochlorite:down-regulationofrobbyMarAandSoxS.ArchMicrobiol,194,933.

10.   ZhangJ,WangX,YangX.(2012)ColorimetricdeterminationofhypochloritewithunmodifiedgoldnanoparticlesthroughtheoxidationofastABIlizerthiolcompound.Analyst,137,2806.


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