![AAT Bioquest/DiBAC4（5）[双-（1,3-二丁基巴比妥酸）五甲基恶唑]/21410/25 mg](https://www.ebiomall.cn/images/AAT201712/chemical-structure-for-dibac4-5-bis-1-3-dibutylbarbituric-acid-pentamethine-oxonol.png)
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 590/616 |
| MW | 542.67 |
| CAS# | 63560-89-4 |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | IonChannels MembranePotentials |
| Related | NeurologicalStains |
| Spectrum | AdvancedSpectrumViewer |
1.Preparecells:
1.1 Foradherentcells:Platecellsovernightingrowthmediumat40,000to80,000cells/well/100µLfor96-wellplatesor10,000to20,000cells/well/25µLfor384-wellplates.
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletsinequalamountofHHBSandMPdye-loADIngsolution(seeStep2.2below)at125,000to250,000cells/well/100µLfor96-wellpoly-Dlysineplatesor30,000to60,000cells/well/25µLfor384-wellpoly-Dlysineplates.Centrifugetheplatesat800rpmfor2minuteswithbrakeoffbeforetheexperiments.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityfortheintracellularcalciummobilization.
2.PrepareDiSBAC2(3)dye-loadingsolution(for1plate):
2.1 Preparea10to30mMstocksolutionofDiSBAC2(3)inhigh-quality,anhydrousDMSO.Thestocksolutionshouldbeusedpromptly;anyremainingsolutionneedbealiquotedandfrozenat<-20oC.
Note:Avoidrepeatedfreeze-thawcycles,andprotectfromlight.
2.2 Preparea2XDiSBAC2(3)dye-loadingsolution:Onthedayoftheexperiment,eitherdissolveDiSBAC2(3)solidinDMSOorthawanaliquotoftheDiSBAC2(3)stocksolutiontoroomtemperature.Preparea2Xworkingsolutionof20to40µMinHanksand20mMHepesbuffer(HHBS)orbufferofyourchoice,pH7with0.04%to0.08%Pluronic®F-127(Cat.#20053)and2mMTrypanRedPlus™(Cat.#2456).Mixthemwellbyvotexing.Thisworkingsolutionisstableforatleast2hoursatroomtemperature.
3.RunMembranePotentialAssay:
3.1 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)DiSBAC2(3)dye-loadingsolution(fromStep2.2)intothecellplate.
Note1:Ifyourscreencompoundsinterferewithgrowthmediumandserumfactors,replacethegrowthmediumwithequalvolumeofHHBSbufferbeforeaddingtheDiSBAC2(3)dye-loadingsolution.Alternatively,cellscanbegrowninserum-freeconditions.
Note2:DoNOTwashthecellsafterdyeloading.
3.2 Incubatethedye-loadingplateinacellincubatorfor30to60minutes.
Note:Insomecases,incubationatroomtemperaturefor30to60minmayworkbetter.
3.3 PreparethecompoundplatesbyusingHHBSoryourdesiredbuffer.
3.4 RunthemembranepotentialassaybymonitoringthefluorescenceintensityatEx/Em=540/590nm(Cat.# 21414).
Note:Itisimportanttorunthesignaltestbeforeyourexperiment.Differentinstrumentshavetheirownintensityrange.Adjustthesignaltestintensitytothelevelof10%to15%ofthemaximuminstrumentintensitycounts.Forexample,themaximumfluorescenceintensitycountforFLIPR-384is65,000,sotheinstrumentsettingsshouldbeadjustedtohaveitssignaltestintensityaround7,000to10,000.
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