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AAT Bioquest/Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit *Green Fluorescence*/11503/200 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 492/515 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | EnzymeDetection HorserADIshPeroxidase(HRP) |
| Related | ReactiveOxygenSpecies MicroplateReaders BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Preparestocksolutions:
1.1 Amplite™GreenPeroxideSensorstocksolution(250X):Add50µLofDMSO(ComponentD)intothevialofAmplite™GreenPeroxideSensor(ComponentA).Thestocksolutionshouldbeusedpromptly.Anyremainingsolutionshouldbealiquotedandrefrozenat-20oC.
Note:Avoidrepeatedfreeze-thawcyclesandprotectfromlight.
1.2 20mMH2O2stocksolution:Add22.7µLof3%H2O2(0.88M,ComponentB)into977µLofAssayBuffer(ComponentC).
Note:ThedilutedH2O2solutionisnotstable.Theunusedportionshouldbediscarded.
2.Prepare1XAmplite™GreenPeroxideSensorworkingsolution:
Add20µLofAmplite™GreenPeroxideSensorstocksolution(250X,fromStep1.1)into5mLofAssayBuffer(ComponentC).
3.PrepareseriallydilutedH2O2standards(0to1000μM):
3.1 Add50μLof20mMH2O2solution(fromStep1.2)into950μLofAssayBuffer(ComponentC)toget1000μMH2O2solution.
3.2 Take200μLof1000μMH2O2solutiontoperform1:3serialdilutionstoget300,100,30,10,3,1,0.3and0μMseriallydilutedH2O2stands.
3.3 AddH2O2standardsandH2O2-containingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables1and2.
Table1.LayoutofH2O2standardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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HS1 | HS1 | …. | …. | …. | …. |
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HS2 | HS2 |
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HS3 | HS3 |
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HS4 | HS4 |
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HS5 | HS5 |
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HS6 | HS6 |
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HS7 | HS7 |
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Note:HS=H2O2Standards;BL=BlankControl;TS=TestSamples
Table2.Reagentcompositionforeachwell
H2O2Standards | BlankControl | TestSample |
SerialDilutions*:50μL | AssayBuffer(ComponentC):50μL | 50μL |
4.RunH2O2assayinsupernatantsreaction:
4.1 Add50μLof1XAmplite™GreenPeroxideSensorworkingsolution(fromStep2)toeachwelloftheH2O2standard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalH2O2assayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLof1XAmplite™GreenperoxideSensorworkingsolutionintoeachwell.
4.2 Incubatethereactionatroomtemperaturefor15to30minutes,protectedfromlight.
4.3 MonitorthefluorescenceincreaseatEx/Em=490±10/520±10nm(optimalEx/Em=490/520)withafluorescenceplatereader.
5.RunH2O2assayinlivecells:
Amplite™GreenPeroxideSensorcanbeloadedpassivelyintolivingcellsandreportthemicromolarchangesinintracellularH2O2concentrations.Thefollowingisasuggestedmicroscopeimagingprotocolthatcanbemodifiedtomeetspecificresearchneeds.
5.1 Activatethecellsasdesired.
5.2 WashthecellswithPBSbuffer,incubatedthecellswith100µL/well1XAmplite™GreenPeroxideSensorworkingsolution(fromStep2)for5to60minutesoryourdesiredtime.
Note:Fora384-wellplate,add25μL/wellof1XAmplite™GreenPeroxideSensorworkingsolution.
5.3 Monitorthefluorescenceincreaseatexcitation490nmandemissionat525nmusingafluorescenceplatereaderwithbottomreadmode.OrimagethefluorescencechangewithafluorescencemicroscopeusingFITCchannel.
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Journal:PLoSOne(2013):e55068
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Authors:He-PingMa
Journal:JournalofBiologicalChemistry(2011):32444--32453
