| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 503/528 |
| MW | 808.69 |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | CellBIOLOGy pHandIonIndicators |
| Related | CellMetabolism BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Preparecellsasdesired.Forexample,plateadherentcellsovernightingrowthmediumat40,000to80,000cells/well/100µLfor
96-wellor10,000to20,000cells/well/25µLfor384-wellplates.
Note: Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
2.PrepareBCECF,AMdye-loADIngsolution:
2.1 Preparea2to20mMstocksolutionofBCECF,AMinhigh-quality,anhydrousDMSO.Thestocksolutionshouldbeusedpromptly.Anyunusedsolutionneedtobealiquotedandrefrozenat<-20oC.
Note:Avoidrepeatedfreeze-thawcycles,andprotectfromlight.
2.2 Preparea5-50uMBCECF,AMdye-loadingsolutioninHanksand20mMHepesbuffer(HHBS).
3. RunpHAssay
3.1 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)BCECF,AMdye-loadingsolutionintothecellplate(fromStep2.3).
Note: ItisimportanttoreplacethegrowthmediumwithHHBSbuffer(100µL/wellfor96-wellplateor25µL/wellfor384-wellplatebeforedye-loading)ifyourcompoundsinterferewiththeserum.
3.2 Incubatethedye-loadingplateatcellincubatorfor30to60minutes.
3.3 Washandreplacethedye-loadingsolutionwithHHBS.
3.4 PreparethecompoundplatesbyusingHHBSoryourdesiredbuffer.
3.5 RunthepHassaybymonitoringthefluorescenceatEx/Em=490/535nm(cutoffat515nm)or505/535nmand430/535nm(cutoffat515nm)forratiomeasurements.Thecompoundadditionis50µL/well(96-wellplate)or25µL/well(384-wellplate).
Note:Theassayshouldbecompletewithin3to5minaftercompoundaddition,howeveraminimumof8mindatacollectionarerecommendedforduringassaydevelopment.
| References&Citations |  CitationExplorer |
TheF0F1ATPsynthaseregulateshumanneutrophilmigrationthroughcytoplasmicprotonextrusioncoupledwithATPgeneration
Authors:JunGao,TianZhang,ZhanfangKang,WeijenTing,LingqingXu,DazhongYin
Journal:MolecularImmunology(2017):219--226
OxidativeStress-ActivatedNHE1IsInvolvedinHighGlucose-InducedApoptosisinRenalTubularEpithelialCells
Authors:YiqingWu,MinZhang,RuiLiu,ChunjieZhao
Journal:YonseiMedicalJournal(2016):1252--1259
