| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 575/590 |
| 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 100XAmplite™Redperoxidasesubstratestocksolution:Add250µLofDMSO(ComponentE)intothevialofAmplite™RedSubstrate(ComponentA).Thestocksolutionshouldbeusedpromptly;anyremainingsolutionshouldbealiquotedandrefrozenat-20oC.
Note:Avoidrepeatedfreeze-thawcyclesandprotectfromlight.
1.2 20U/mLPeroxidasestocksolution:Add1mLofAssayBuffer(ComponentC)intothevialofHorseradishPeroxidase(ComponentD).
Note:TheunusedHRPsolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
1.3 20mMH2O2stocksolution:Add22.7µLof3%H2O2(0.88M,ComponentB)into977µLofAssayBuffer(ComponentC).
Note:ThedilutedH2O2solutionisnotstable.Theunusedportionshouldbediscarded.
2.PrepareH2O2reactionmixture:
PreparetheH2O2reactionmixtureaccordingtothefollowingtableandkeepfromlight:
Table1H2O2Reactionmixtureforone96-wellplate(2X)
Components | Volume |
Amplite™RedPeroxidaseSubstrateStockSolution(100X,fromStep1.1) | 50µL |
20U/mlPeroxidaseStockSolution(fromStep1.2) | 200µL |
AssayBuffer(ComponentC) | 4.75mL |
Totalvolume | 5mL |
3.PrepareserialdilutuionsofH2O2standard(0to10μM):
3.1 Add1μLof20mMH2O2solution(fromStep1.3)into1999μLofAssayBuffer(ComponentC)togeta10 μMH2O2standard.
3.2 Take200μLof10μMH2O2standardtoperform1:3serialdilutionstoget3,1,0.3,0.1,0.03,0.01and0 μMserialdilutionsofH2O2standard.
3.3 AddserialdilutionsofH2O2standardandH2O2-containingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables2and3.
Table2LayoutofH2O2standardsandtestsamplesinasolidblack96-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
Table3Reagentcompositionforeachwell
H2O2Standard | BlankControl | TestSample |
Serialdilutions*:50μL | AssayBuffer(ComponentC):50μL | 50μL |
*Note:AddtheseriallydilutedH2O2standardsfrom0.01μMto10μMintowellsfromHS1toHS7induplicate.HighconcentrationofH2O2(e.g.,>100μM,finalconcentration)maycausereducedfluorescencesignalduetotheoveroxidationofAmplite™Red(toanon-fluorescentproduct).
4.RunH2O2assayinsupernatantsreaction:
4.1 Add50μLofH2O2reactionmixture(fromStep2)intoeachwellofH2O2standard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalH2O2assayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofH2O2reactionmixtureineachwell.
4.2 Incubatethereactionatroomtemperaturefor15to30minutes,protectedfromlight.
4.3 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=540±10/590±10nm(optimalEx/Em=540/590nm).
Note:Thecontentsoftheplatecanalsobetransferredtoawhiteclearbottomplateandreadbyanabsorbancemicroplatereaderatthewavelengthof576±5nm.Theabsorptiondetectionhaslowersensitivitycomparedtofluorescencereading.
5.RunH2O2assayforcells:
TheAmplite™FluorimetricHydrogenPeroxideAssayKitcanbeusedtomeasurethereleaseofH2O2fromcells.Thefollowingisasuggestedprotocolthatcanbemodifiedtomeetthespecificresearchneeds.
5.1 TheH2O2reactionmixtureshouldbepreparedasStep2exceptthattheAssayBuffer(ComponentC)shouldbereplacedwiththemediathatisusedinyourcellculturesystem.Suggestedmediaincluding(a)KrebsRingersPhosphateBuffer(KRPB);(b).HanksBalancedSaltSolution(HBSS);or(c)Serum-freemedia.
5.2 Preparecellsina96-wellplate(50-100μL/well),andactivatethecellsasdesired.
Note:Thenegativecontrols(mediaaloneandnon-activatedcells)areincludedformeasuringbackgroundfluorescence.
5.3 Add50μLofH2O2reactionmixture(fromStep5.1)intoeachwellofcellsandH2O2standards(fromStep3.3).
Note:Fora384-wellplate,add25μLofcellsand25μLofH2O2reactionmixtureintoeachwell.
5.4 Incubatethereactionatroomtemperaturefor15to30minutes,protectedfromlight.
5.5 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=540±10/590±10nm(optimalEx/Em=540/590nm).
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Journal:ProcessBiochemistry(2016):1486--1495
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Journal:PLoSOne(2013):e55068
Hydrogenperoxidestimulatestheepithelialsodiumchannelthroughaphosphatidylinositide3-kinase-dependentpathway
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Journal:JournalofBiologicalChemistry(2011):32444--32453
