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AAT Bioquest/DCFH-DA [2',7'-Dichlorodihydrofluorescein diacetate] *CAS 4091-99-0*/15204/25 mg
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| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 504/529 |
| MW | 487.29 |
| CAS# | 4091-99-0 |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | EnzymeDetection RedoxEnzymes |
| Related | CellSignalingMolecules ReactiveOxygenSpecies BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
- Make1-10mMDMSOstocksolution.TheunusedDMSOstocksolutionshouldbealiquotedintoasingleusevialandstoredat-20°C.Keepfromlight.
- Makethedyeworkingconcentrationof1-10µMinaphysiologicalbuffer(suchasPBS,HBSS,HEPES).Theoptimalworkingconcentrationforyourapplicationmustbeempiricallydetermined.
- Removecellsfromgrowthmedia,addthedyeworkingsolution(fromStep2)tothecells,andincubatethecellsatRTor37oCfor5to60minutes.
- Removethedyeworkingsolution;washwithpre-warmedHBSS,andaddpre-warmedHBSSorgrowthmediumandincubateattheoptimaltemperature.Theoptimalrecoverytimecanvarywidely,assomecelltypesnormallyexhibitlowlevelsofesteraseactivity.
- Determinethebaselinefluorescenceintensityofasampleoftheloadedcellspriortoexposingthecellstoexperimentalinducements.
- Negativecontrolsshouldbeassessedasfollows:
6.1Examineunstainedcellsforautofluorescenceinthegreenemissionrange.
6.2Forflowcytometry,ascertainthattheforwardandsidescatterofcellsisunchangedafterdye-loADIngandtreatment.Changesincelldimensionsmayberelatedtoblebbingorshrinkageresultingfromhandlingoratoxicresponse.
6.3Examinethefluorescenceofcell-freemixturesofdyeandbuffer/mediawithandwithouttheinducer.Intheabsenceofextracellularesterasesandotheroxidativeenzymes,thegradualincreaseinfluorescenceovertimemayberelatedtospontaneoushydrolysis,atmosphericoxidation,and/orlight-inducedoxidation.
6.4Examinethefluorescenceofuntreated(control)loadedcellsthathavebeenmaintainedingrowthmediumorsimplebuffer.Inhealthycells,oxygenradicalsareeliminatedbycellularenzymesand/ornaturalantioxidants.Followingthedye-loadingrecoveryperiod,healthycellsshouldexhibitalowleveloffluorescencethatisrelativelystableforthedurationoftheexperiment;however,agradualincrease(duetoauto-oxidation)ordecrease(duetolossofdyefromcellsorphotobleaching)influorescencemaybeobserved.Intheabsenceofanystimulusorinducement,aburstoffluorescenceinhealthy,untreatedcellscouldindicateprogresstocelldeathorsomeotheroxidativeevent.
- PositivecontrolsmaybestimulatedwithH2O2ortert-butylhydroperoxide(TBHP)toafinalconcentrationof~100µM(increaseordecreasedosebasedonthesensitivityandresponseofthecells).
| References&Citations |  PrinterFriendlyVersion |
1. DiazG,LiuS,IsolaR,DianaA,FalchiAM.(2003)Mitochondriallocalizationofreactiveoxygenspeciesbydihydrofluoresceinprobes.HistochemCellBiol,120,319.
2. SaengkhaeC,LoetchutinatC,Garnier-SuillerotA.(2003)Kineticanalysisoffluoresceinanddihydrofluoresceineffluxesintumourcellsexpressingthemultidrugresistanceprotein,MRP1.BiochemPharmacol,65,969.
3. HempelSL,BuettnerGR,O"MalleyYQ,WesselsDA,FlahertyDM.(1999)Dihydrofluoresceindiacetateissuperiorfordetectingintracellularoxidants:comparisonwith2",7"-dichlorodihydrofluoresceindiacetate,5(and6)-carboxy-2",7"-dichlorodihydrofluoresceindiacetate,anddihydrorhodamine123.FreeRadicBiolMed,27,146.
4. SchillingE,DopkeHH.(1978)ArapiddiagnostictestfortheviABIlityofearlycattleandrabbitembryosusingdiacetyl-fluorescin.Naturwissenschaften,65,658.
5. RobertsonEJ,PatelJA.(1968)Determinationoffluorescinsodiuminophthalmicsolutions.AmJHospPharm,25,598.
6. ManciniAM,ZampaGA,CostanziG.(1961)[Experimentalresearchoninsulinantibodies.II.Immunochemicalcharacterizationofguineapiganti-oxinsulinserumconjugatedwithfluorescinisothiocyanate.].BollSocItalBiolSper,37,1230.
7. CostanziG,ManciniUM,ZampaGA.(1961)[Experimentalresearchoninsulinantibodies.III.Immunohistologyoftheendocrinepancreaswithguineapiganti-bovineinsulinserum,conjugatedwithfluorescinisothiocyanate.].BollSocItalBiolSper,37,1233.
8. KingEO,WardMK,RaneyDE.(1954)Twosimplemediaforthedemonstrationofpyocyaninandfluorescin.JLabClinMed,44,301.
9. TotterJR,MoseleyFT.(1953)InfluenceoftheconcentrationofironontheproductionoffluorescinbyPseudomonasaeruginosa.JBacteriol,65,45.
10. HurstL,LowburyEJ.(1952)TransferoffluorescinfromPs.pyocyaneatocoloniesofotherbacteria.JClinPathol,5,359.
