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AAT Bioquest/Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit *Blue Fluorescence*/11504/100 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 405/450 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | EnzymeDetection HorserADIshPeroxidase(HRP) |
| Related | ReactiveOxygenSpecies MicroplateReaders |
1.Preparecells:
1.1. Foradherentcells:Platecellsovernightingrowthmediumat10,000to40,000cells/well/90µLfora96-wellplateor2,500to10,000cells/well/22.5µLfora384-wellplate.
1.2. Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandsUSPendthecellpelletsinculturemediumat50,000-100,000cells/well/90µLfora96-wellpoly-Dlysineplateor10,000-25,000cells/well/22.5µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortoyourexperiment.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
2.PrepareOxiVision™Blueperoxidesensorstocksolution:
2.1 PrepareOxiVision™Blueperoxidesensorstocksolution(500X):Add40µLofDMSO(ComponentC)intothevialofOxiVision™Blueperoxidesensor(ComponentA),andmixthemwell.
Note:20µLofreconstitutedofreconstitutedOxiVision™Blueperoxidesensorstocksolutionisenoughfor1plate.Thestocksolutionshouldbeusedpromptly.Anyremainingsolutionshouldbealiquotedandfrozenat-20oC.Avoidrepeatedfreeze-thawcyclesandprotectfromlight.
2.2 PrepareOxiVision™Blueperoxidesensorworkingsolution(10X):Add10µLof500XDMSOreconstitutedOxiVision™Blueperoxidesensorstocksolution(fromStep2.1)into500µLofAssayBuffer(ComponentB),andmixthemwell.
Note:Theworkingsolutionisnotstable;prepareitasneededbeforeuse.
3.Runthehydrogenperoxideassay:
3.1 Add10µL/well(96-wellplate)ofOxiVision™Blueperoxidesensorworkingsolution(fromStep2.2)in90µLcellcultureperwellinthecellplate.
Note1:Itisnotnecessarytowashcellsbeforestaining.It"srecommendedtostainthecellsinfullmedium.
Note2:Fora384-wellplate,add2.5µL/wellof1XOxiVision™Blueperoxidesensorworkingsolution
3.2 Treatcellswithtestcompoundsinfullmediumorinyourdesiredbufferat37°Cfordesiredperiodoftime.Forcontrolsamples(untreatedcells),addthecorrespondingamountofcompoundbuffer.
Note1:It"srecommendedtotreatcellsinfullmedium.However,iftestedcompoundsareserumsensitive,growthmediumandserumfactorscanbeaspiratedawaybeforetreatment.Add1XHank"ssaltsolutionand20mMHepesbuffer(HHBS)orthebufferofyourchoiceintothecellsafteraspiration.Alternatively,cellscanbetreatedinserum-freemedia.
Note2:WetreatedJurkatcellswith100µMhydrogenperoxideinfullmediumat37°Cfor90minutestoinducehydrogenperoxide.SeeFigure1fordetails.
3.3 WashcellswithDPBS1-2times,andreplacewith100uL/well(for96-wellplate)or25uL/well(for384-wellplate)AssayBuffer(ComponentC).TakeimagesusingfluorescencemicroscopewithaDAPIfilter.
| References&Citations |  CitationExplorer |
Plasma-activatedmediumselectivelyeliminatesundifferentiatedhumaninducedpluripotentstemcells
Authors:RyoMatsumoto,KazunoriShimizu,TakunoriNagashima,HiromasaTanaka,MasaakiMizuno,FumitakaKikkawa,MasaruHori,HiroyukiHonda
Journal:RegenerativeTherapy(2016):55--63
