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Brand移液器移液枪移液器吸头容量瓶瓶口分液器
来自 : mayitao
Reactivity
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Application
BiochemicalAssay(BCA)
Options
Bulkdiscount
Supplier
SupplierProductNo.
PurposeTheOxiSelect™IntracellularROSAssayKitisacell-basedassayformeasuringantioxidantorROSactivity.Cellsareculturedina96-wellcellcultureplateandthenpre-incubatedwithDCFH-DA,whichiscell-permeable.TheunknownantioxidantorROSsamplesarethenaddedtothecells.Afterabriefincubation,thecellscanbereadonastandardfluorescenceplatereader.TheROSorantioxidantcontentinunknownsamplesisdeterminedbycomparisonwiththepredeterminedDCFstandardcurve.
BrandOxiSelect™
SampleTypeCellSamples
DetectionMethodFluorometric
CharacteristicsOxiSelect™IntracellularROSAssayKit(GreenFluorescence)isacell-basedassayformeasuringhydroxyl,peroxyl,orotherreactiveoxygenspeciesactivitywithinacell.Theassayemploysthecell-permeablefluorogenicprobe2,7-Dichlorodihydrofluorescindiacetate(DCFH-DA).Inbrief,DCFH-DAisdiffusedintocellsandisdeacetylatedbycellularesterasestonon-fluorescent2,7-Dichlorodihydrofluorescin(DCFH),whichisrapidlyoxidizedtohighlyfluorescent2,7-Dichlorodihydrofluorescein(DCF)byROS(Figure1).ThefluorescenceintensityisproportionaltotheROSlevelswithinthecellcytosol.TheeffectofantioxidantorfreeradicalcompoundsonDCF-DAcanbemeasuredagainstthefluorescenceoftheprovidedDCFstandard.ThekithasaDCFdetectionsensitivitylimitof10pM.Eachkitprovidessufficientreagentstoperformupto96assays,includingstandardcurveandunknownsamples.
Components
  1. 20XDCFH-DA:One500μLambertubeofa20mMsolutioninmethanol.
  2. DCFStandard:One100μLambertubeofa1mMsolutioninDMSO.
  3. HydrogenPeroxide:One100μLambertubeofan8.821Msolution.
  4. 2XCellLysisBuffer:One20mLbottle.3
Materialnotincluded
  1. SterileDPBSforwashesandbufferdilutions
  2. HanksBalancedSaltSolution(HBSS)
  3. Cellculturemedium(ie:DMEM+/-10%FBS)
  4. 96-wellblackorfluorescencemicrotiterplate
  5. Fluorescentmicroplatereadercapableofreading480nm(excitation)and530nm(emission)
BackgroundAccumulationofreactiveoxygenspecies(ROS)coupledwithanincreaseinoxidativestresshasbeenimplicatedinthepathogenesisofseveraldiseasestates.Theroleofoxidativestressinvasculardiseases,diabetes,renalischemia,atherosclerosis,pulmonarypathologicalstates,inflammatorydiseases,andcancerhasbeenwellestablished.Freeradicalsandotherreactivespeciesareconstantlygeneratedinvivoandcauseoxidativedamagetobiomolecules,aprocessheldincheckbytheexistenceofmultipleantioxidantandrepairsystemsaswellasthereplacementofdamagednucleicacids,proteinsandlipids.MeasuringtheeffectofantioxidanttherapiesandROSactivityintracellularlyiscrucialtosuppressingortreatingoxidativestressinducers.
ApplicationNotesOptimalworkingdilutionshouldbedeterminedbytheinvestigator.
Comment

  • Quick~1hourprotocol
  • Highlysensitiveto10pM
  • DetectsthepresenceofvariousROSspecies

AssayTime1h
ReagentPreparation
  • 1XDCFH-DA:Dilutethe20XDCFH-DAstocksolutionto1Xincellculturemedia,preferablywithoutFBS.Stirorvortextohomogeneity.Prepareonlyenoughforimmediateapplications.Notes:
  • 1XDCFH-DA/mediasolutioncontains5 %methanol.Forcellsthataresensitivetomethanol,werecommendinsteadpreparinga0.1X(100μM)solutionofDCFH-DAincellculturemedia.
  • Duetolight-inducedauto-oxidation,DCFH-DAsolutionsatanyconcentrationmustbeprotectedfromlight.
  • HydrogenPeroxide(H2O2):PrepareH2O2dilutionsinDMEMorDPBSasnecessary.Donotstoredilutedsolutions.HydrogenPeroxidemaybeusedasapositivecontrolintheassay,orasacelltreatment.
AssayProcedure

I.DCFDyeLoading

  1. Prepareandmixallreagentsthoroughlybeforeuse.Eachunknownsampleshouldbeassayedinduplicateortriplicate.
  2. Culturecellsineitheraclearorblack96-wellcellcultureplate.Note:Ifusingablackplate,chooseanappropriateplatebasedonyourfluorometersreader(i.e.chooseaclearbottomblackplateforbottomreaders).
  3. Removemediafromallwellsanddiscard.WashcellsgentlywithDPBSorHBSS2-3times.Removethelastwashanddiscard.
  4. Add100μLof1XDCFH-DA/mediasolutiontocells.Incubateat37 °Cfor30-60 minutes.
  5. Removesolution.RepeatstepthreeusingmultiplewasheswithDPBSorHBSS.Removethelastwashanddiscard.
  6. TreatDCFH-DAloadedcellswithdesiredoxidantorantioxidantin100μLmedium.II.QuantitationofFluorescence•FluorescencemicroscopyorFlowcytometry:Fluorescencecanbeanalyzedonaninvertedfluorescencemicroscopeorbyflowcytometryusingexcitationandemissionwavelengthsof480nmand530nm,respectively.•FluorescencePlateReader:•Assaysperformedinblackcellculturefluorometricplates:Platemaybereadimmediatelyforkineticanalysisorafter1hourforstaticanalysis.Platesreadforkineticanalysismaybereadinincrementsof1and5 minutesupto1hourormoreasnecessary.Readthefluorescencewithafluorometricplatereaderat480nm/530nm.•Assaysperformedinclearcellcultureplates:Aftertreatmentwithdesiredoxidantorantioxidant,carefullyremovetreatmentmediafromallwellsanddiscard.WashcellsgentlywithDPBSorHBSS2-3times.Removethelastwashanddiscard.Add100μLofmediumtoeachwell.Add100μLofthe2XCellLysisBuffer,mixthoroughlyandincubate5 minutes.Transfer150μLofthemixturetoa96-wellplatesuitableforfluorescencemeasurement.Readthefluorescencewithafluorometricplatereaderat480nm/530nm.

RestrictionsForResearchUseonly
HandlingAdviceAvoidmultiplefreeze/thawcycles.
Storage4°C/-20°C
StorageCommentUponreceipt,storetheDCFH-DAandDCFStandardat-20°C.Avoidmultiplefreeze/thawcycles.StoretheCellLysisBufferandHydrogenPeroxideat4°C.
SupplierImages
Cellular Assay (CA) image for OxiSelect™ Intracellular ROS Assay Kit (Green Fluorescence) (ABIN2344999)ROSinHeLaCellsTreatedwithHydrogenPeroxide. 50,000HeLacellsina96-wellplat...
Productcitedin:Yao,Zou,Wang,Ji,Yang:"PinoresinolDiglucosideAlleviatesoxLDL-InducedDysfunctioninHumanUmbilicalVeinEndothelialCells."in:Evidence-basedcomplementaryandalternativemedicine:eCAM,Vol.2016,pp.3124519,2017(PubMed).

Golestaneh,Chu,Xiao,Stoleru,Theos:"DysfunctionalautophagyinRPE,acontributingfactorinage-relatedmaculardegeneration."in:Celldeath&disease,Vol.8,Issue1,pp.e2537,2017(PubMed).

Crookenden,Walker,Heiser,Murray,Dukkipati,Kay,Meier,Moyes,Mitchell,Loor,Roche:"Effectsofprecalvingbodyconditionandprepartumfeedinglevelongeneexpressionincirculatingneutrophils."in:Journalofdairyscience,Vol.100,Issue3,pp.2310-2322,2017(PubMed).

Zhang,Zhao,Sun,Li,Wei,Ashman,Hu:"DifferentvirulenceofcandidaalbicansisattributedtotheabilityofescapefromneutrophilextracellulartrapsbysecretionofDNase."in:Americanjournaloftranslationalresearch,Vol.9,Issue1,pp.50-62,2017(PubMed).

Ludwig,Teran,Teichgraeber,Hilger:"Nanoparticle-basedhyperthermiadistinctlyimpactsproductionofROS,expressionofKi-67,TOP2A,andTPX2,andinductionofapoptosisinpancreaticcancer."in:Internationaljournalofnanomedicine,Vol.12,pp.1009-1018,2017(PubMed).

Vijayarathna,Oon,Chen,Kanwar,Sasidharan:"PolyalthialongifoliaMethanolicLeafExtracts(PLME)induceapoptosis,cellcyclearrestandmitochondrialpotentialdepolarizationbypossiblymodulatingtheredoxstatusinhelacells."in:Biomedicine&pharmacotherapy,Vol.89,pp.499-514,2017(PubMed).

Kavitha,EinOon,Chen,Kanwar,Sasidharan:"Phaleriamacrocarpa(Boerl.)fruitinduceG0/G1andG2/Mcellcyclearrestandapoptosisthroughmitochondria-mediatedpathwayinMDA-MB-231humanbreastcancercell."in:Journalofethnopharmacology,Vol.201,pp.42-55,2017(PubMed).

Xu,Kong:"Bixinameliorateshighfatdiet-inducedcardiacinjuryinmicethroughinflammationandoxidativestresssuppression."in:Biomedicine&pharmacotherapy,Vol.89,pp.991-1004,2017(PubMed).

Yu,Pan,Dong,Niu:"AstragalosideIVattenuatesleadacetate-inducedinhibitionofneuriteoutgrowththroughactivationofAkt-dependentNrf2pathwayinvitro."in:Biochimicaetbiophysicaacta,Vol.1863,Issue6,pp.1195-1203,2017(PubMed).

Domey,Bergemann,Bremer-Streck,Krumbein,Reichenbach,Teichgräber,Hilger:"Long-termprevalenceofNIRF-labeledmagneticnanoparticlesforthediagnosticandintraoperativeimagingofinflammation."in:Nanotoxicology,Vol.10,Issue1,pp.20-31,2016(PubMed).

Wang,Sun,Chen,Jiao,Bai:"ROS-mediatedactivationofJNK/p38contributespartiallytothepro-apoptoticeffectofajoeneoncellsoflungadenocarcinoma."in:Tumourbiology,2016(PubMed).

Slusarczyk,Trojan,Glombik,Piotrowska,Budziszewska,Kubera,Popiolek-Barczyk,Lason,Mika,Basta-Kaim:"Anti-inflammatorypropertiesoftianeptineonlipopolysaccharide-inducedchangesinmicroglialcellsinvolvetoll-likereceptor-relatedpathways."in:Journalofneurochemistry,Vol.136,Issue5,pp.958-70,2016(PubMed).

Marondedze,Wong,Thomas,Irving,Gehring:"CyclicNucleotideMonophosphatesinPlantsandPlantSignaling."in:Handbookofexperimentalpharmacology,2016(PubMed).

Kuranaga,Yamada,Kashiwaya,Nakamura,Cui,Kumazaki,Shinohara,Sugito,Taniguchi,Ito,Nakayama,Uno,Itoh,Akao:"Anti-Oncogenicgem-DihydroperoxidesInduceApoptosisinCancerCellsbyTrappingReactiveOxygenSpecies."in:Internationaljournalofmolecularsciences,Vol.17,Issue1,2016(PubMed).

Yamaguchi,Madhyastha,Madhyastha,Choijookhuu,Hishikawa,Pengjam,Nakajima,Maruyama:"Arsenicacidinhibitsproliferationofskinfibroblasts,andincreasescellularsenescencethroughROSmediatedMST1-FOXOsignalingpathway."in:TheJournaloftoxicologicalsciences,Vol.41,Issue1,pp.105-13,2016(PubMed).

Johnson,Dludla,Joubert,February,Mazibuko,Ghoor,Muller,Louw:"Aspalathin,adihydrochalconeC-glucoside,protectsH9c2cardiomyocytesagainsthighglucose-inducedshiftsinsubstratepreferenceandapoptosis."in:Molecularnutrition&foodresearch,2016(PubMed).

Liu,Chen,Xue,Zhao,Di:"Mefloquineeffectivelytargetsgastriccancercellsthroughphosphatase-dependentinhibitionofPI3K/Akt/mTORsignalingpathway."in:Biochemicalandbiophysicalresearchcommunications,Vol.470,Issue2,pp.350-5,2016(PubMed).

Tu,Zhang,Wei,Li,Zhang,Yang,Xing:"AllicinattenuatesH2O2?inducedcytotoxicityinretinalpigmentedepithelialcellsbyregulatingthelevelsofreactiveoxygenspecies."in:Molecularmedicinereports,2016(PubMed).

Kim,Beaven,Kulinski,Desai,Bandara,Bai,Prussin,Schwartz,Komarow,Metcalfe,Olivera:"RegulationofReactiveOxygenSpeciesandtheAntioxidantProteinDJ-1inMastocytosis."in:PLoSONE,Vol.11,Issue9,pp.e0162831,2016(PubMed).
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发布于 ： 2018-03-25 阅读（94）
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