| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 494/517 |
| MW | 913.05 |
| CAS# | N/A |
| Solvent | Water |
| Storage | F/D/L |
| Category | GPCR CalciumGPCRAssays |
| Related | CalciumChannels pHandIonIndicators BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
UseofFluo-8®AMEsters
1.LoadCellswithFluo-8®AMEsters:
AMestersarethenon-polarestersthatreadilycrosslivecellmembranes,andrapidlyhydrolyzedbycellularesterasesinsidelivecells.AMestersarewidelyusedforloadingavarietyofpolarfluorescentprobesintolivecellnon-invasively.However,cautionsmustbeexcisedwhenAMestersareusedsincetheyaresusceptibletohydrolysis,particularlyinsolution.Theyshouldbereconstitutedjustbeforeuseinhigh-quality,anhydrousdimethylsulfoxide(DMSO).DMSOstocksolutionsmaybestoreddesiccatedat–20°Candprotectedfromlight.Undertheseconditions,AMestersshouldbestableforseveralmonths.
FollowingisourrecommendedprotocolforloadingFluo-8®AMestersintolivecells.Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.
a) Preparea2to5mMstocksolutionofFluo-8®AMestersinhigh-quality,anhydrousDMSO.
b) Onthedayoftheexperiment,eitherdissolveFluo-8®inDMSOorthawanaliquotoftheindicatorstocksolutiontoroomtemperature.Prepareaworkingsolutionof1to10µMinHanksandHepesbuffer(HHBS)orthebufferofyourchoicewith0.02%Pluronic®F-127.Formostofcelllines,Fluo-8®reagentswithaconcentrationrangingfrom4-5uMarerecommended.Theexactconcentrationoftheindicatorrequiredforcellloadingmustbedeterminedempirically.Toavoidanyartifactscausedbyoverloadingandpotentialdyetoxicity,itisrecommendedtousetheminimaldyeconcentrationthatcangeneratesufficientsignalstrength.
Note:ThenonionicdetergentPluronic®F-127issometimesusedtoincreasetheaqueoussolubilityofFluo-8®AMesters. AvarietyofPluronic®F-127solutionscanbepurchasedfromAATBioquest.
c) Ifyourcellscontainingtheorganicanion-transports,probenecid(1–2.5mM)orsulfinpyrazone(0.1–0.25mM)maybeaddedtothecellmediumtoreduceleakageofthede-esterifiedindicators.
Note:AvarietyofReadiUse™probenecidincludingwatersolublesodiumsaltandstABIlizedsolutioncanbepurchasedfromAATBioquest.
d) Addequalvolumeofthedyeworkingsolution(fromStepborc)intoyourcellplate.
e) Incubatethedye-loadingplateatacellincubatororroomtemperaturefor20minutestoonehour.
Note:Decreasingtheloadingtemperaturemightreducethecompartmentalizationoftheindictor.
f) ReplacethedyeworkingsolutionwithHHBSorbufferofyourchoice(containingananiontransporterinhibitor,suchas2.5mMprobenecid,ifapplicable)toremoveexcessprobes.
g) RuntheexperimentsatEx/Em =490/525nm
UseofScreenQuest™Fluo-8NWCalciumAssayKitsforHTSApplications
GPCRactivationcanbedetectedbydirectmeasurementofthereceptormediatedcAMPaccumulation,orchangesinintracellularCa2+concentration.GPCRtargetsthatcoupleviaGqproduceanincreaseinintracellularCa2+thatcanbemeasuredusingacombinationofFluo-8®reagentsandafluorescencemicroplatereader.Thefluorescenceimagingplatereaders(suchas,FLIPR™,FDSSorBMGNovoStar™)haveacooledCCDcameraimagingsystemwhichcollectsthesignalfromeachwellofamicroplate(both96and384-well)simultaneously.Theseplatereaderscanreadatsub-secondintervals,whichenablesthekineticsoftheresponsetobecaptured,andhasanintegratedpipettorthatmaybeprogrammedforsuccessiveliquidadditions.BesidestheirrobustapplicationsforGPCRtargets,ourScreenQuest™Fluo-8CalciumAssayKitscanbealsousedforcharacterizingcalciumionchannelsandscreeningcalciumionchannel-targetedcompounds.
Figure2.CarbacholDoseResponsewasmeasuredinHEK-293cellswithScreenQuest™Fluo-8NWAssaykitandFluo-4NWAssayKit.HEK-293cellswereseededovernightat40,000cells/100µL/wellina96-wellblackwall/clearbottomcostarplate.Thegrowthmediumwasremoved,andthecellswereincubatedwith,respectively,100µLoftheScreenQuest™Fluo8-NWcalciumassaykitandFluo-4NWkit(accordingtothemanufacturer’sinstructions)for1houratroomtemperature.Carbachol(25µL/well)wasaddedbyNOVOstar(BMGLabTech)toachievethefinalindicatedconcentrations.TheEC50ofFluo-8NWisabout1.2uM.
ComparedtoothercommercialcalciumassaykitsthateitherbasedonFluo-3orFluo-4,ourScreenQuest™CalciumAssayKitshavethefollowingadvantagesforHTSapplications:
•BroadApplications:workwithbothGPCRandcalciumchanneltargets.
•ConvenientSpectralWavelengths:maximumexcitation@~490nm;maximumemission@~514nm.
•FlexibleDyeLoading:dyeloadingatroomtemperature(ratherthan37ºCrequiredforFluo-4AM).
•NoWashRequiredandNoQuencherInterferencewithYourTargets.
•RobustPerformance:enablecalciumassaysthatareimpossiblewithFluo-4AMorFluo-3AM.
•StrongestSignalIntensity:2timesbrighterthanthatofFluo-4AM;4timesbrighterthanthatofFluo-3AM.
UseofFluo-8®Salts
Calciumcalibrationcanbecarriedoutbymeasuringthefluorescenceintensityofthesaltform(25to50µMinfluorescencemicroplatereaders)oftheindicatorsinsolutionswithpreciselyknownfreeCa2+concentrations.Calibrationsolutionscanbeusedbasedon30mMMOPSEGTACa2+buffer.Ingeneral,watercontainstraceamountofcalciumion.Itishighlyrecommendedtouse30mMMOPS+100mMKCl,pH7.2asbuffersystem.Onecansimplymakea0and39µMcalciumstocksolutionsaslistedbelow,andthese2solutionsareusedtomakeaserialsolutionofdifferentCa2+concentrations
A.0µMcalcium:30mMMOPS+100mMKCl,pH7.2buffer+10mMEGTA
B.39µMcalcium:30mMMOPS+100mMKCl,pH7.2buffer+10mMEGTA+10mMCaCl2
TodetermineeitherthefreecalciumconcentrationofasolutionortheKdofasingle-wavelengthcalciumindicator,thefollowingequationisused:
[Ca]free=Kd[F─Fmin]/Fmax─F]
WhereFisthefluorescenceintensityoftheindicatorataspecificexperimentalcalciumlevel,FministhefluorescenceintensityintheabsenceofcalciumandFmaxisthefluorescenceintensityofthecalcium-saturatedprobe.
Thedissociationconstant(Kd)isameasureoftheaffinityoftheprobeforcalcium.Thecalcium-bindingandspectroscopicpropertiesoffluorescentindicatorsvaryquitesignificantlyincellularenvironmentscomparedtocalibrationsolutions.InsituresponsecalibrationsofintracellularindicatorstypicallyyieldKdvaluessignificantlyhigherthaninvitrodeterminations.InsitucalibrationsareperformedbyexposingloadedcellstocontrolledCa2+buffersinthepresenceofionophoressuchasA-23187,4-bromoA-23187andionomycin.Alternatively,cellpermeabilizationagentssuchasdigitoninorTriton®X-100canbeusedtoexposetheindicatortothecontrolledCa2+levelsoftheextracellularmedium.TheKdvaluesofFluo-8®reagentsarelistedinTable1foryourreference.
| References&Citations |  CitationExplorer |
Below,youmayfindasmallsamplingofspecificFluo-8®AMapplicationssortedbyfieldofstudy.ToinquireaboutapotentialapplicationofFluo-8®AM,ortoconsultwithourfluorescentdyespecialists,pleasecontactusatsupport@aatbio.comor1-800-990-8053.
InOncology,Fluo-8®AMhasbeenusedtostudy:
»BreastcancercellsbymonitoringintracellularCa2+fluxassociatedwithapoptosisandinhibitionby2-aminoethoxydiphenylborate[1]
»Antitumoractivitybywayofthioredoxin-bindingprotein2anditsdependenceonintracellularcalciumconcentration[2]
»Bcl-1andBcl-2regulationthroughcharacterizationofcytosolictransportasquantifiedbycalciumflux[3]
»Ca2+influxandCa2+channelactivityinNCI-H460cellsasaparameterformonitoringprogressionofnon-smallcelllungcancer[4]
»Ca2+releasebyHN4cellsandCLIC4upregulationofapoptosisthroughmitochondrialandendoplasmicreticulumpathways[5]
InCardiology,Fluo-8®AMhasbeenusedtostudy:
»Low-energyfar-fieldstimulationasatherapyfortachycardiaandfibrillation[6]
»Calciumfluxduringcalciumsparksinventricularmyocytes[7]
»Cardiacconductionasafunctionofcellrigidityinthecontextofcardiovasculardisease[8]
»DiastolicCa2+transientsincardiacmyocytesandSR-luminalandfreecytoplasmicCa2+concentrations[9]
»Sphingosine-1-phosphate(S1P)receptoractivationinvalvularinterstitialcellsasdetectedbycytosolicCa2+flux[10]
InNeurobiology,Fluo-8®AMhasbeenusedtostudy:
»HippocampalCA1neurons,visualizatingneuronstoinvestigatetheroleofamyloid-βintheprogressionofAlzheimer"sdisease[11]
»CytosolicCa2+concentrationsinHEK293cellsanditsregulatoryeffectonAβ1-42andhAmylinandassociatedsignalingpathways[12]
»Gprotein-coupledreceptors(GPRs)inresponsetocannabinoidsinpresynapticCA3orpostsynapticCA1pyramidalcells[13]
»Medullaryinterneuronsanddendriticcalciumactivityinregardstoinspiratorybursts[14]
»N2acellactivationbyhistamine,asmonitoredbyincreasesinintracellularCa2+concentrations[15]
InStemCells,Development&Differentiation,Fluo-8®AMhasbeenusedtostudy:
»Inductionofpluripotentstemcells(iPSCs)intofunctioningcardiaccells,asvalidatedbyCa2+fluxandmembranepotential[16]
»CXCR4andCXCR7receptorsinTcellsandtheirroleincellsurvivalandchemotaxis[17]
»Ca2+uptakebymyocytesderivedfromhumaninducedpluripotentstemcellsduringpathogenesisofDuchennemusculardystrophy[18]
»AgoNIST-inducedcalciumtransientsindifferentiationofratbonemarrowmesenchymalstemcellsintosmoothmusclecells[19]
»CalciumchannelblockadesandtheireffectoncardiacProgenitorcellproliferationanddifferentiation[20]
AAT Bioquest常用产品报价单
| 产品名 | 货号 | 公司分类 | 商城分类 | 美金价 |
| AAT Bioquest/Fluorescein, disodium salt *CAS 518-47-8*/2/100 mg | 2 | iFluor Rapid Tests | 酸碱缓冲液 | 1088.00 | AAT Bioquest/14-3-3 ζ (Ab-58) Antibody/8B0001/50 ug | 8B0001 | Signaling Intermediate Ab | 重组抗体 | 2828.00 | AAT Bioquest/Opioid Receptor (Phospho-Ser375) Antibody/8A0022/50 ug | 8A0022 | Phospho-Specific Ab | 受体 | 2828.00 | AAT Bioquest/iFluor™ 350 goat anti-mouse IgG (H+L)/16440/200 ug | 16440 | Fluorescent Anti-IgGs | 荧光染料 | 1088.00 | AAT Bioquest/trFluor™ Eu goat anti-mouse IgG (H+L)/16518/100 ug | 16518 | Fluorescent Anti-IgGs | 抗小鼠 | 4278.00 | AAT Bioquest/RPE-streptavidin conjugate/16900/100 ug | 16900 | Streptavidin Conjugates | 其他生物染料 | 1378.00 | AAT Bioquest/GCNT3 Antibody/8C14708/50 ug | 8C14708 | Signaling Intermediate Ab | 功能性抗体 | 2828.00 | AAT Bioquest/Amplite™ Luciferase Reporter Gene Assay Kit *Bright Glow*/12518/1 plate | 12518 | Reporter Gene Enzymes | 其它检测试剂盒 | 2103.00 | AAT Bioquest/ReadiLink™ Rapid mFluor™ Violet 450 Antibody Labeling Kit *Microscale Optimized for Labeling 50 &mi | 1100 | mFluor™ Dyes and Kits | 标记试剂盒 | 2103.00 | AAT Bioquest/Amplite™ Colorimetric Urea Quantitation Kit *Blue Color*/10058/200 Tests | 10058 | Diagnostic Molecules | 定量试剂盒 | 2828.00 | AAT Bioquest/Screen Quest™ CHO-Gqi Chimera Cell line/38101/Each | 38101 | cAMP GPCR Assays | 常规细胞株 | 0.00 |
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