| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 571/585 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy CellMetabolism |
| Related | RedoxEnzymes BiochemicalAssays |
1.Preparestocksolutions:
1.1 Amplite™Redsubstratesolution(250X):Add40µLofDMSO(ComponentF)intothevialofAmplite™Redsubstrate(ComponentA).Thestocksolutionshouldbeusedpromptly.Anyremainingsolutionshouldbealiquotedandrefrozenat-20°C.
Note1:Avoidrepeatedfreeze-thawcycles.
Note2:Amplite™Redsubstrateisunstableinthepresenceofthiolssuchasdithiothreitol(DTT)and2-mercaptoethanol.ThefinalconcentrationofDTTor2-mercaptoethanolinthereactionshouldbenohigherthan10μM.TheassayshouldbeperformedatpH7–8(pH7.4isrecommended)asAmplite™RedisunstablepH>8.5.
1.2 HRPstocksolution(500X):Add100µLofassaybuffer(ComponentB)intothevialofhorserADIshperoxidase(ComponentC).
Note:TheunusedHRPsolutionshouldbedividedintosingleusealiquotsandstoredat-20°C.
1.3 10U/mLxanthineoxidase(XO)solution:Add100µLofassaybuffer(ComponentB)intothevialofxanthineoxidase(ComponentE)tomake10U/mLxanthineoxidase(XO)solution.
Note:Theunusedxanthineoxidasesolutionshouldbedividedintosingleusealiquotsandstoredat-20°C.
2. Prepareassaymixture:
Prepareassaymixtureaccordingtothefollowingtablesandprotectfromlight.
Table1.Assaymixtureforone96-wellplate(2X):
Components | Volume |
Amplite™RedSubstrateStockSolution(250X,fromStep1.1) | 20µL |
HRP(500X,fromStep1.2) | 10µL |
XanthineOxidase(100X,ComponentE) | 50µL |
AssayBuffer(ComponentB) | 5mL |
Totalvolume | 5.08mL |
3.Prepareseriallydilutedxanthinestandards(0to100µM):
3.1 Add5μLofXanthineStandard(ComponentD)into995μLofAssayBuffer(ComponentB)toget100µMxanthinestandardsolution.
3.2 Take200μLof100µMxanthinestandardsolutiontoperform1:3serialdilutionstoget100,30,10,3,1,0.3,0.1and0µMseriallydilutedxanthinestandards.
3.3 Add50μLofxanthinestandardsand/orxanthine-containingtestsamplesintoablackwall/solidbottom96-wellmicroplateasdescribedinTables2.
Table2.LayoutofXanthinstandardsandtestsamplesinablackwall/solidbottom96-wellmicroplate(50μL/well).
BL | BL | TS | TS | …. | …. |
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X1 | X1 | …. | …. | …. | …. |
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X2 | X2 |
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X3 | X3 |
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X4 | X4 |
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X5 | X5 |
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X6 | X6 |
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X7 | X7 |
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Note1:X=xanthinestandards,BL=blankcontrol,TS=testsamples.Note2:AddtheseriallydilutedXanthinestandardsfrom0.137μMto100μMintoeachwellfromX1toX7induplicate.Note3:HighconcentrationofxanthinemaycausereducedfluorescencesignalduetotheoveroxidationofAmplite™Redsubstrate(toanon-fluorescentproduct).
4.Runxanthineassay:
4.1 Add50μLofassaymixture(fromStep2)intoeachwellofthexanthinestandards,blankcontrol,andtestsamples(seeStep3,Table2)tomakethetotalxanthineassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofassaymixtureintoeachwell.
4.2 Incubatethereactionfor30to60minutesatroomtemperature,protectedfromlight.
4.3 MonitortheabsorbanceincreasewithwithafluorescenceplatereaderatEx/Em=530-570nm/590-600nm(optimalEx/Em=540/590nm),cutoff=570nm.
| References&Citations |  PrinterFriendlyVersion |
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2. MartinDE,DeAlmeidaJF,HenryMA,KhaingZZ,SchmidtCE,TeixeiraFB,DiogenesA.(2014)Concentration-dependenteffectofsodiumhypochloriteonstemcellsofapicalpapillasurvivalanddifferentiation.JEndod,40,51.
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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.
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