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AAT Bioquest/Screen Quest™ Fluo-8 Medium Removal Calcium Assay Kit *Optimized for Difficult Cell Lines*/36307/1 Plate
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 490/525 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | GPCR CalciumGPCRAssays |
| Related | CalciumChannels pHandIonIndicators |
| Spectrum | AdvancedSpectrumViewer |
Warning:Donotaddadditionalprobenecid.ItisrecommendedtoincubatethedyeloADIngsolutionnolongerthan2hours.
1.Preparecells:
1.1 Foradherentcells:Platecellsovernightingrowthmediumat40,000to80,000cells/well/100µLfora96-wellplateor10,000to20,000cells/well/25µLfora384-wellplate.
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletinFluo-8NWdye-loadingsolution(seeStep2.4)at125,000to250,000cells/well/100µLfora96-wellpoly-Dlysineplateor30,000to60,000cells/well/25µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiments.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityfortheintracellularcalciummobilization.
2.PrepareFluo-8NWdye-loadingsolution(foroneplate):
2.1 Thaw1vialofFluo-8NW(ComponentA),1bottleof10XPluronic®F127Plus(ComponentB)andHHBS(ComponentC)atroomtemperaturebeforeuse.
2.2 MakeFluo-8NWstocksolution:Add10µL(forCat.#36307)or100µL(forCat.#36308and#36309)ofDMSOintoFluo-8NW(ComponentA),andmixthemwell.
Note:10µLofFluo-8NWstocksolutionisenoughfor1plate.UnusedFluo-8NWstocksolutioncanbealiquotedandstoredat<-20oCformorethanonemonthifthetubesaresealedtightly.Protectfromlightandavoidrepeatedfreeze-thawcycles.
2.3 Make1Xassaybuffer:
a).ForCat.#36307(1platekit)and36308(10plateskit),make1Xassaybufferbyadding9mLofHHBS(ComponentC)into10XPluronic®F127Plus(1mL,ComponentB),andmixthemwell.
b).ForCat.#36309(100plateskit),make1Xassaybufferbyaddingthewholebottleof10XPluronic®F127Plus(10mL,ComponentB)into90mLofHHBSbuffer(notincludedinthekit),andmixthemwell.
Note:10mLof1Xassaybufferisenoughforoneplate.Aliquotandstoreunused1Xassaybufferat<‑20 oC.Protectfromlightandavoidrepeatedfreeze-thawcycles.
2.4 MakeFluo-8NWdye-loadingsolutionforonecellplate:Add10µLofFluo-8NWDMSOstocksolution(fromStep2.2)into10mLof1Xassaybuffer(fromStep2.3),andmixthemwell.Thisworkingsolutionisstableforatleast2hoursatroomtemperature.
3.Runcalciumassay:
3.1 Removethegrowthmediumfromthecellplate.
Note1:Itisimportanttoremovethegrowthmediuminordertominimizebackgroundfluorescence,andcompoundinterferencewithserumorculturemedia.
Note2:Alternatively,growthecellsingrowthmediumwith0.5-to1%FBStoavoidmediumremovalstep.Inthiscase,2XdyeloadingsolutioninHHBSbufferisneeded.[Weoffer2separatenowashcalciumassaykits(Cat.#36315andCat.#36316)forthosewhouse0.5-to1%FBSingrowthmediumtoavoidthemediumremovalstep].
3.2 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofFluo-8NWdye-loadingsolution(fromStep2.4)intothecellplate(fromStep3.1).
3.3 Incubatethedye-loadingplateinacellincubatorfor30minutes,andthenincubatetheplateatroomtemperatureforanother30minutes.
Note1:Iftheassayrequires37oC,performtheexperimentimmediatelywithoutfurtherroomtemperatureincubation.
Note2:Ifthecellscanfunctionwellatroomtemperatureforlongertime,incubatethecellplateatroomtemperaturefor1-2hours(Itisrecommendedthattheincubationtimebenolongerthan2hours.)
3.4 PreparethecompoundplateswithHHBSoryourdesiredbuffer.
3.5 RunthecalciumfluxassaybymonitoringthefluorescenceintensityatEx/Em=490/525nm.
Note:Itisimportanttorunthesignaltestbeforeyourexperiment.Differentinstrumentshavetheirownintensityrange.Adjustthesignaltestintensitytothelevelof10%to15%ofthemaximumintensitycounts.Forexample,themaximumfluorescenceintensitycountforFLIPR-384is65,000,sotheinstrumentsettingshouldbeadjustedtohaveitssignaltestintensityaround7,000to10,000.
| 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]
