| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 458/597 |
| MW | 808.98 |
| CAS# | N/A |
| Solvent | Water |
| Storage | F/D/L |
| Category | GPCR CalciumGPCRAssays |
| Related | CalciumChannels pHandIonIndicators BiochemicalAssays |
| 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 |
1. WendtER,FerryH,GreavesDR,KeshavS.(2015)Ratiometricanalysisoffuraredbyflowcytometry:atechniqueformonitoringintracellularcalciumfluxinprimarycellsubsets.PLoSOne,10,e0119532.
2. BaileyS,MacardlePJ.(2006)AflowcytometriccomparisonofIndo-1tofluo-3andFuraRedexcitedwithlowpowerlasersfordetectingCa(2+)flux.JImmunolMethods,311,220.
3. SuZL,LiN,SunYR,YangJ,WangIM,JiangSC.(1998)[Monitoringcalciuminouterhaircellswithconfocalmicroscopyandfluorescenceratiosoffluo-3andfura-red].ShiYanShengWuXueBao,31,323.
4. WalczyskoP,WagnerE,AlbrechtovaJT.(2000)Useofco-loadedFluo-3andFuraRedfluorescentindicatorsforstudyingthecytosolicCa(2+)concentrationsdistributioninlivingplanttissue.CellCalcium,28,23.
5. WuY,ClusinWT.(1997)Calciumtransientalternansinblood-perfusedischemichearts:observationswithfluorescentindicatorfurared.AmJPhysiol,273,H2161.
6. BlackwoodAM,SagnellaGA,MarkanduND,MacGregorGA.(1997)ProblemsassociatedwithusingFura-2tomeasurefreeintracellularcalciumconcentrationsinhumanredbloodcells.JHumHypertens,11,601.
7. FlotoRA,Mahaut-SmithMP,SomasundaramB,AllenJM.(1995)IgG-inducedCa2+oscillationsindifferentiatedU937cells;astudyusinglaserscanningconfocalmicroscopyandco-loadedfluo-3andfura-redfluorescentprobes.CellCalcium,18,377.
8. NovakEJ,RabinovitchPS.(1994)Improvedsensitivityinflowcytometricintracellularionizedcalciummeasurementusingfluo-3/FuraRedfluorescenceratios.Cytometry,17,135.
9. SchildD,JungA,SchultensHA.(1994)LocalizationofcalciumentrythroughcalciumchannelsinolfactoryreceptorneuronesusingalaserscanningmicroscopeandthecalciumindicatordyesFluo-3andFura-Red.CellCalcium,15,341.
10. KurebayashiN,HarkinsAB,BaylorSM.(1993)Useoffuraredasanintracellularcalciumindicatorinfrogskeletalmusclefibers.BiophysJ,64,1934.
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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