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AAT Bioquest/OG488 BAPTA-1, AM [equivalent to Oregon Green® 488 BAPTA-1, AM] *Cell permeant*/20507/10x50 ug
Overview | PrinterFriendlyVersion |
Ex/Em(nm) | 494/523 |
MW | 1258.07 |
CAS# | N/A |
Solvent | DMSO |
Storage | F/D/L |
Category | GPCR CalciumGPCRAssays |
Related | CalciumChannels pHandIonIndicators |
Spectrum | AdvancedSpectrumViewer |
UseofCalciumindicatorAMEsters
1.LoadCellswithCalciumIndicatorAMEsters:
AMestersarethenon-polarestersthatreADIlycrosslivecellmembranes,andrapidlyhydrolyzedbycellularesterasesinsidelivecells.AMestersarewidelyusedforloadingavarietyofpolarfluorescentprobesintolivecellnon-invasively.However,cautionsmustbeexcisedwhenAMestersareusedsincetheyaresusceptibletohydrolysis,particularlyinsolution.Theyshouldbereconstitutedinhigh-quality,anhydrousdimethylsulfoxide(DMSO).DMSOstocksolutionsshouldbestoreddesiccatedat-20°Candprotectedfromlight.Undertheseconditions,AMestersshouldbestableforseveralmonths.
FollowingisourrecommendedprotocolforloadingAMestersintolivecells.Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.
a) Preparea2to5mMAMestersstocksolutioninhigh-quality,anhydrousDMSO.
b) Onthedayoftheexperiment,eitherdissolvecalciumindicatorssolidinDMSOorthawanaliquotoftheindicatorstocksolutionstoroomtemperature.Prepareaworkingsolutionof2to20µMinthebufferofyourchoice(suchasHanksandHepesbuffer)with0.04%Pluronic®F-127.Formostcelllineswerecommendthefinalconcentrationofcalciumindicatorsbe4-5uM.Theexactconcentrationofindicatorsrequiredforcellloadingmustbedeterminedempirically.Toavoidanyartifactscausedbyoverloadingandpotentialdyetoxicity,itisrecommendedtousetheminimalprobeconcentrationthatcanyieldsufficientsignalstrength.
Note:ThenonionicdetergentPluronic®F-127issometimesusedtoincreasetheaqueoussolubilityofcalciumindicatorAMesters. AvarietyofPluronic®F-127solutionscanbepurchasedfromAATBioquest.
c) Ifyourcells(suchasCHOcells)containingtheorganicanion-transports,probenecid(2–5mM)orsulfinpyrazone(0.2–0.5mM)maybeaddedtothethedyeworkingsolution(finalinwellconcentrationwillbe1-2.5mMforprobenecid,or0.1-0.25mMforsulfinpyrazone)toreducetheleakageofthede-esterifiedindicators.
Note:AvarietyofReadiUse™probenecidincludingwatersolublesodiumsaltandstABIlizedsolutioncanbepurchasedfromAATBioquest
d) Addequalvolumeofthedyeworkingsolution(fromStepborc)intoyourcellplate.
e) Incubatethedye-loadingplateroomattemperatureor37°Cfor20minutes(especiallyFluo-8AM)to2hours,andthenincubatetheplateatroomtemperatureforanother30minutes.
Note1:Decreasingtheloadingtemperaturemightreducethecompartmentalizationoftheindictor.
Note2:IncubatetheCal-520AMlongerthan2hoursgivesbettersignalintensityforsomecelllines.
f) ReplacethedyeworkingsolutionwithHHBSorbufferofyourchoice(containingananiontransporterinhibitor,suchas1mMprobenecid,ifapplicable)toremoveexcessprobes.
g) RuntheexperimentsatdesiredEx/Emwavelengths(seeTable1).
2.MeasureIntracellularCalciumResponses:
Figure1.ResponseofendogenousP2YreceptortoATPinCHO-M1cellswithoutprobenecid.CHO-M1cellswereseededovernightat40,000cellsper100µLperwellina96-wellblackwall/clearbottomcostarplate.100µlof4µMFluo-3AM,Fluo-4AMorCal520®AMinHHBSwereaddedintothewells,andthecellswereincubatedat37°Cfor2hour.Thedyeloadingmediumwerereplacedwith100µlHHBS,50µlof300µMATPwereadded,andthenimagedwithafluorescencemicroscope(OlympusIX71)usingFITCchannel.
A B
Figure2.ATP-stimulatedcalciumresponseofendogenousP2YreceptorinCHO-K1cellsmeasuredwithCal-520®orFluo-4AM.CHO-K1cellswereseededovernightin50,000cellsper100µLperwellina96-wellblackwall/clearbottomcostarplate.100µLof5µMFluo-4AMortheCal-520®AMwith(A)orwithout(B)2.5mMprobenecidwasaddedintothecells,andthecellswereincubatedat37oCfor2hours. ATP(50µL/well)wasaddedbyFlexStation(MolecularDevices)toachievethefinalindicatedconcentrations.
UseofCalciumindicatorSalts
TodetermineeitherthefreecalciumconcentrationofasolutionortheKdofasingle-wavelengthcalciumindicator,thefollowingequationisused:
[Ca]free=Kd[F-Fmin]/Fmax-F]
WhereFisthefluorescenceoftheindicatoratexperimentalcalciumlevels,FministhefluorescenceintheabsenceofcalciumandFmaxisthefluorescenceofthecalcium-saturatedprobe.Thedissociationconstant(Kd)isameasureoftheaffinityoftheprobeforcalcium.TheCa2+-bindingandspectroscopicpropertiesoffluorescentindicatorsvaryquitesignificantlyincellularenvironmentscomparedtocalibrationsolutions.InsitucalibrationsofintracellularindicatorstypicallyyieldKdvaluessignificantlyhigherthaninvitrodeterminations.InsitucalibrationsareperformedbyexposingloadedcellstocontrolledCa2+buffersinthepresenceofionophoressuchasA-23187,4-bromoA-23187andionomycin.Alternatively,cellpermeabilizationagentssuchasdigitoninorTriton®X-100canbeusedtoexposetheindicatortothecontrolledCa2+levelsoftheextracellularmedium.TheKdvaluesofsomecalciumreagentsarelistedinTable1foryourreference.
UseofCalciumindicatorConjugates
Comparedtothefreeionindicator,dextranconjugatesofthesesameindicatorsexhibitbothreducedcompartmentalizationandmuchlowerratesofdyeleakage.Sincethemolecularweightofthedextran,netcharge,degreeoflabeling,andnatureofthedyemayaffecttheexperiment,researchersareadvisedtoconsulttheprimaryliteratureforinformationspecifictotheapplicationofinterest.
References&Citations | PrinterFriendlyVersion |
- SchnellC,NegmM,DriehausJ,SchellerA,HulsmannS.(2016)Norepinephrine-inducedcalciumsignalinginastrocytesintherespiratorynetworkoftheventrolateralmedulla.RespirPhysiolNeurobiol,226,18.
- DanielAG,LaffontP,ZhaoM,MaH,SchwartzTH.(2015)Opticalelectrocorticogram(OECoG)usingwide-fieldcalciumimagingrevealsthedivergenceofneuronalandglialactivityduringacuterodentseizures.EpilepsyBehav,49,61.
- TadaM,TakeuchiA,HashizumeM,KitamuraK,KanoM.(2014)Ahighlysensitivefluorescentindicatordyeforcalciumimagingofneuralactivityinvitroandinvivo.EurJNeurosci,39,1720.
- TomekJ,NovakO,SykaJ.(2013)Two-PhotonProcessorandSeNeCA:afreelyavailablesoftwarepackagetoprocessdatafromtwo-photoncalciumimagingatspeedsdowntoseveralmillisecondsperframe.JNeurophysiol,110,243.
- SchmitzJ,HogerU,TorkkeliPH,FrenchAS.(2012)Calciumbufferingandclearanceinspidermechanosensoryneurons.JCompPhysiolANeuroetholSensNeuralBehavPhysiol,198,477.
- TakataN,MishimaT,HisatsuneC,NagaiT,EbisuiE,MikoshibaK,HiraseH.(2011)Astrocytecalciumsignalingtransformscholinergicmodulationtocorticalplasticityinvivo.JNeurosci,31,18155.
- LangerD,HelmchenF.(2012)PosthocimmunostainingofGABAergicneuronalsubtypesfollowinginvivotwo-photoncalciumimaginginmouseneocortex.PflugersArch,463,339.
- LattaruloC,ThyssenD,KuchibholtaKV,HymanBT,BacskaiqBJ.(2011)Microscopicimagingofintracellularcalciuminlivecellsusinglifetime-basedratiometricmeasurementsofOregonGreenBAPTA-1.MethodsMolBiol,793,377.
- WotzlawC,BernardiniA,Berchner-PfannschmidtU,PapkovskyD,AckerH,FandreyJ.(2011)Multifocalanimatedimagingofchangesincellularoxygenandcalciumconcentrationsandmembranepotentialwithintheintactadultmousecarotidbodyexvivo.AmJPhysiolCellPhysiol,301,C266.
- BouhoursB,TrigoFF,MartyA.(2011)SomaticdepolarizationenhancesGABAreleaseincerebellarinterneuronsviaacalcium/proteinkinaseCpathway.JNeurosci,31,5804.