| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | None/None |
| MW | 764.68 |
| CAS# | 126150-97-8 |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | GPCR CalciumGPCRAssays |
| Related | CalciumChannels pHandIonIndicators BiochemicalAssays |
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 |  CitationExplorer |
HelicobacterpyloriinducesIL-1βproteinthroughtheinflammasomeactivationindifferentiatedmacrophagiccells
Authors:ShoichiroKameoka,TakeshiKameyama,TakayaHayashi,SeiichiSato,NaomiOhnishi,TakeruHayashi,NaokoMurata-Kamiya,HideakiHigashi,MasanoriHatakeyama,AkinoriTakaoka
Journal:BiomedicalResearch(2016):21--27
LicochalconeAinducesT24bladdercancercellapoptosisbyincreasingintracellularcalciumlevels
Authors:XinhuiYang,JiangtaoJiang,XinyanYang,JichunHan,QiushengZheng
Journal:Molecularmedicinereports(2016):911--919
Spautin-1AmelioratesAcutePancreatitisviaInhibitingImpairedAutophagyandAlleviatingCalciumOverload
Authors:JuanXiao,XuepingFeng,Xiao-YingHuang,ZhongshiHuang,YanqiangHuang,ChaoganLi,GenliangLi,SongNong,RuoshiWu,YongzhiHuang
Journal:MolecularMedicine(2016):643
DelayedadmiNISTrationofthenucleicacidanalog2Cl-C.OXT-Aattenuatesbraindamageandenhancesfunctionalrecoveryafterischemicstroke
Authors:NaohikoOkabe,EmiNakamura,NaoyukiHimi,KazuhikoNarita,IkukoTsukamoto,TokumiMaruyama,NorikazuSakakibara,TakehiroNakamura,ToshifumiItano,OsamuMiyamoto
Journal:Brainresearch(2013):115--131
Rasguanylnucleotidereleasingprotein2affectscellviabilityandcell--matrixadhesioninECV304endothelialcells
Authors:JunichiTakino,KentaroNagamine,TakamitsuHori
Journal:Celladhesion&migration(2013):262--266
Involvementofthioredoxin-bindingprotein2intheantitumoractivityofCD437
Authors:SaoriMatsuoka,HiroyukiTsuchiya,TomohikoSakabe,YumiWatanabe,YoshikoHoshikawa,AkihiroKurimasa,HiroakiItamochi,TasukuHarada,NaokiTerakawa,HiroshiMasutani
Journal:Cancerscience(2008):2485--2490
