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AAT Bioquest/Screen Quest™ Fura-2 No Wash Calcium Assay Kit/36320/10 Plates
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 370/476 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | GPCR CalciumGPCRAssays |
| Related | CalciumChannels pHandIonIndicators BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.PrepareCells:
1.1 Foradherentcells:Platecellsovernightingrowthmediumat40,000to80,000cells/well/100µLfora96-wellplateor10,000to20,000cells/well/25µLfora384-wellplate.
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletsinequalamountofHHBSandFura-2AMdye-loadingsolution(seesteps2.4below)at125,000to250,000cells/well/100µLfora96-wellpoly-Dlysineplateor30,000to60,000cells/well/25µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiments.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityfortheintracellularcalciummobilization.
2.PrepareFura-2AMdye-loadingsolution:
2.1 Thaw1vialofFura-2AM(ComponentA),1bottleof10XPluronicF127Plus(ComponentB)andHHBS(ComponentC)atroomtemperaturebeforeuse.
2.2 MakeFura-2AMstocksolution:Add200µLofDMSOintothevialofFura-2AM(ComponentA),andmixthemwell.
Note:20µLofreconstitutedFura-2AMstocksolutionisenoughforoneplate.UnusedreconstitutedFura-2AMstocksolutioncanbealiquotedandstoredat<-20oCforuptothreemonthsifthetubesaresealedtightlyandkeptfromlight.Avoidrepeatedfreeze-thawcycles.
2.3 Make1Xassaybuffer:
a).ForCat#36320(10plateskit),make1Xassaybufferbyadding9mLofHHBS(ComponentC)intothebottleof10XPluronicF127Plus(ComponentB,1mL),andmixthemwell.
b).ForCat#36321(100plateskit),make1Xassaybufferbyaddingthewholebottleof10XPluronicF127Plus(ComponentB,10mL)into90mLofHHBSbuffer(notincludedinthekit),andmixthemwell.
Note:10mLof1Xassaybufferisenoughfor1plate.Aliquotandstoreun-used1Xassaybufferat<-20oCandkeptfromlight.Avoidrepeatedfreeze-thawcycles.
2.4 MakeFura-2AMdye-loadingsolutionforonecellplate:Add20µLofDMSOreconstitutedFura-2AMstocksolution(fromStep2.2)into10mLof1Xassaybuffer(fromStep2.3),andmixthemwell.Thisdye-loadingsolutionisstableforatleast2hoursatroomtemperature.
3.RunCalciumAssay:
3.1 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofFura-2AMdye-loadingsolutionintothecellplate.
Note:Ifyourcompoundsinterferewiththeserum,itisimportanttoreplacethegrowthmediumwithHHBSbuffer(100µL/wellfora96-wellplateor25µL/wellfora384-wellplatebeforedye-loading).
3.2 Incubatethedye-loadingplateatcellincubatorfor1hour,andthenincubatetheplateatroomtemperatureforanother20minutes.
Note1:Ifassaysrequire37oC,performtheexperimentimmediatelywithoutfurtherroomtemperatureincubation.
3.3 PreparethecompoundplatebyusingHHBSoryourdesiredbuffer.
3.4 RunthecalciumfluxassaybymonitoringthefluorescenceincreaseatEx/Em=340/510nmand380/510nmasdescribedintheinstrumentmanuals.
ForassaysperformedonaFlexStation,usethefollowingwavelengthparameters:
Excitation Emission Cutoff
Lm1340 510nm 455nm
Lm2380 510nm 455nm
ForassaysperformedonanFDSS,usethestandardfiltersforFura-2calciumassaysontheinstrument.
Note.Dispensespeedandheightforcompoundadditionsneedtobeoptimizedforeachassay.
| References&Citations |  PrinterFriendlyVersion |
1. Barreto-ChangOL,DolmetschRE.(2009)CalciumimagingofcorticalneuronsusingFura-2AM.JVisExp.
2. HirstRA,HarrisonC,HirotaK,LambertDG.(2006)Measurementof[Ca2+]iinwholecellsuspensionsusingfura-2.MethodsMolBiol,312,37.
3. ShaoM,WangHM,LiuZH,ShenP,CaiRX.(2005)[LoadofcalciumprobeFura-2/AMinEscherichiacolicells].WeiShengWuXueBao,45,805.
4. FroschauerEM,KolisekM,DieterichF,SchweigelM,SchweyenRJ.(2004)Fluorescencemeasurementsoffree[Mg2+]byuseofmag-fura2inSalmonellaenterica.FEMSMicrobiolLett,237,49.
5. McConnellG,RiisE.(2004)Photoniccrystalfibreenablesshort-wavelengthtwo-photonlaserscanningfluorescencemicroscopywithfura-2.PhysMedBiol,49,4757.
6. XuT,YangW,HuoXL,SongT.(2004)AbnormalspectraalterationobservedinTritoncalibrationmethodformeasuring[Ca2+]iwithfluorescenceindicator,fura-2.JBiochemBiophysMethods,58,219.
7. BerkovaZ,MorrisAP,EstesMK.(2003)Cytoplasmiccalciummeasurementinrotavirusenterotoxin-enhancedgreenfluorescentprotein(NSP4-EGFP)expressingcellsloadedwithFura-2.CellCalcium,34,55.
8. FischerW,FrankeH,ScheiblerP,AllgaierC,IllesP.(2002)AMPA-inducedCa(2+)influxinculturedratcorticalnonpyramidalneurones:pharmacologicalcharacterizationusingfura-2microfluorimetry.EurJPharmacol,438,53.
9. NovakI,NitschkeR,AmstrupJ.(2002)Purinergicreceptorshavedifferenteffectsinratexocrinepancreas.Calciumsignalsmonitoredbyfura-2usingconfocalmicroscopy.CellPhysiolBiochem,12,83.
10. Billing-MarczakK,PrzybyszewskaM,KuznickiJ.(1999)Measurementsof[Ca2+]usingfura-2ingliomaC6cellsexpressingcalretininwithGFPasaMarkeroftransfection:noCa2+-bufferingprovidedbycalretinin.BiochimBiophysActa,1449,169.
