| 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.PrepareOxirite™HypochloriteSensorstocksolution(200X):
Add50µLofDMSOintothevialofOxirite™HypochloriteSensor(ComponentA)tomake200Xstocksolution.
Note:Makeasingleusealiquots,andstoreunused200XOxirite™HypochloriteSensorstocksolutionat-20ºC,avoidlightandrepeatfreeze-thawcycles.
2.Preparehypochloriteassaymixture:
Add25µLofOxirite™HypochloriteSensorstocksolution(fromStep1)into5mLofAssayBuffer(ComponentB),andmixwelltomakehypochloriteassaymixture(ComponentA+B).
Note1:Thishypochloriteassaymixtureisenoughforone96-wellplate.Itisnotstable,useitpromptly.
3.PrepareserialdilutionsofHypochloriteStandards(0to100mµ):
3.1 Preparehypochloritestandarddilutions:Add50μLofhypochloritestandard(ComponentC)into450μLofAssayBuffer(ComponentB)toget100mMhypochloritestandardsolution.Thenperform1:10and1:3serialdilutionsinAssayBuffer(ComponentB)togetapproximately10,1,0.1,0.01,0.001,0.003and0mMseriallydilutedhypochloritestandards.
Note:Theunusedhypochloritestandard(ComponentC)shouldbedividedintosingleusealiquotsandstoredat-20ºC.
3.2 Addserialdilutionsofhypochloritestandardandhypochlorite/hypochlorousacidcontainingtestsamplesintoa96-wellsolidblackmicroplateasdescribedinTables1and2.
Table1Layoutofhypochloritestandardsandtestsamplesina96-wellsolidblackmicroplate
BL | BL | TS | TS | …. | …. |
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H1 | H1 | …. | …. | …. | …. |
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H2 | H2 |
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H3 | H3 |
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H4 | H4 |
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H5 | H5 |
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H6 | H6 |
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H7 | H7 |
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Note:H=HypochloriteStandards,BL=BlankControl,TS=TestSamples.
Table2Reagentcompositionforeachwell
HypochlorousAcidStandard | BlankControl | TestSample |
SerialDilutions*:50μL | AssayBuffer:50μL | 50μL |
*Note:Addtheseriallydilutedhypochloritestandardsfrom0.003mMto100mMintowellsfromH1toH7induplicate.
4.Runhypochlorite/hypochlorousacidassay:
4.1 Add50μLofhypochloriteassaymixture(fromStep2)toeachwellofhypochloritestandards,blankcontrol,andtestsamples(seeStep3.2)tomakethetotalvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofhypochloriteassaymixtureintoeachwell.
4.2 Incubatethereactionatroomtemperaturefor10-30minutes,protectedfromlight.
4.3 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=540/590nm,cutoff570nm.
| References&Citations |  PrinterFriendlyVersion |
1. KitamuraK,MaruyamaK,HamanoS,KishiT,KawakamiT,TakahashiY,OnoderaS.(2014)Effectofhypochloriteoxidationoncholinesterase-inhibitionassayofacetonitrileextractsfromfruitsandvegetablesformonitoringtracesoforganophosphatepesticides.JToxicolSci,39,71.
2. MartinDE,DeAlmeidaJF,HenryMA,KhaingZZ,SchmidtCE,TeixeiraFB,DiogenesA.(2014)Concentration-dependenteffectofsodiumhypochloriteonstemcellsofapicalpapillasurvivalanddifferentiation.JEndod,40,51.
3. YinB,DengJ,PengX,LongQ,ZhaoJ,LuQ,ChenQ,LiH,TangH,ZhangY,YaoS.(2013)Greensynthesisofcarbondotswithdown-andup-conversionfluorescentpropertiesforsensitivedetectionofhypochloritewithadual-readoutassay.Analyst,138,6551.
4. TakimotoK,TaharaguchiM,SakaiK,TakagiH,TohyaY,YamadaYK.(2013)Effectofhypochlorite-baseddisinfectantsoninactivationofmurinenorovirusandattempttoeliminateorpreventinfectioninmicebyadditiontodrinkingwater.ExpAnim,62,237.
5. SetlowB,YuJ,LiYQ,SetlowP.(2013)AnalysisofthegerminationkineticsofindividualBacillussubtilissporestreatedwithhydrogenperoxideorsodiumhypochlorite.LettApplMicrobiol,57,259.
6. ThornRM,RobinsonGM,ReynoldsDM.(2013)Comparativeantimicrobialactivitiesofaerosolizedsodiumhypochlorite,chlorinedioxide,andelectRochemicallyactivatedsolutionsevaluatedusinganovelstandardizedassay.AntimicrobAgentsChemother,57,2216.
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.
10. ZhangJ,WangX,YangX.(2012)ColorimetricdeterminationofhypochloritewithunmodifiedgoldnanoparticlesthroughtheoxidationofastABIlizerthiolcompound.Analyst,137,2806.
