Overview | PrinterFriendlyVersion |
Ex/Em(nm) | 490/514 |
MW | ~600 |
CAS# | N/A |
Solvent | DMSO |
Storage | F/D/L |
Category | NeuroBIOLOGy ReactiveOxygenSpecies |
Related | CellFunctionalAnalysis RedoxEnzymes BiochemicalAssays |
Spectrum | AdvancedSpectrumViewer |
1.PrepareOxiVisionGreenTMhydrogenperoxidesensorworkingsolution:
1.1 Preparea2to5mMstocksolutionofOxiVisionGreenTMhydrogenperoxidesensorinhigh-quality,anhydrousDMSO.Thestocksolutionshouldbeusedpromptly;anyremainingsolutionshouldbealiquotedandfrozenat-20oC.
Note:Avoidrepeatedfreeze-thawcycles.
1.2 Preparea2XOxiVisionGreenTMhydrogenperoxidesensorworkingsolution:Onthedayoftheexperiment,eitherdissolveOxiVisionGreenTMhydrogenperoxidesensorsolidinDMSOorthawanaliquotofthesensorstocksolutiontoroomtemperature.Preparea2Xworkingsolutionattheconcentrationrangingfrom2to20µMin20mMHepesbufferorbufferofyourchoice,pH7.ItisrecommendedtouseOxiVisionGreenTMhydrogenperoxidesensoratthefinalconcentrationof5µMtomeasureH2O2concentrationinsolution.
2.RunH2O2Assayinsupernatants:
2.1 Add50µLof2XOxiVisionGreenTMhydrogenperoxidesensorworkingsolution(fromStep1.2)toeachwelloftheH2O2standard,blankcontrol,andtestsamplestomakethetotalH2O2assayvolumeof100 µL/well.
Note:Fora384-wellplate,add25µLofsampleand25µLof2XOxiVisionGreenTMhydrogenperoxidesensorworkingsolutionintoeachwell.
2.2 Incubatethereactionatroomtemperaturefor15to60minutes,protectedfromlight.
2.3 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=490/525nm.
2.4 Thefluorescenceinblankwells(withtheassaybufferonly)isusedasacontrol,andissubtractedfromthevaluesforthosewellswiththeH2O2reactions.
3.RunH2O2AssayinLiveCells:
OxiVisionGreenTMhydrogenperoxidesensorcanbeloadedpassivelyintolivingcellsandreportthemicromolarchangesinintracellularH2O2concentrations.Thefollowingisasuggestedmicroscopeimagingprotocolwhichcanbemodifiedaccordingtoyourspecificresearchneeds.
3.1 TheOxiVisionGreenTMhydrogenperoxidesensorworkingsolutionshouldbepreparedasStep1.2.ItisrecommendedtousePBSorHanksBalancedSaltSolution(HBSS)with20mMHepesbufferinsteadof20mMHepesbufferonly.
3.2 Treatthecellsasdesired.
3.3 IncubatethecellswithOxiVisionGreenTMhydrogenperoxidesensorworkingsolutionfor5to60minoradesiredperiodoftime.WashthecellswithPBSbuffertwice.
3.4 MonitorthefluorescenceincreaseatEx/Em=490/525nmwithafluorescenceplatereaderwithbottomreadmode.OrimagethefluorescencechangebyafluorescencemicroscopyusingtheFITCchannel.
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