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Ex/Em(nm)	503/528
MW	~600
CAS#	N/A
Solvent	DMSO
Storage	F/D/L
Category	CellBIOLOGy pHandIonIndicators
Related	CellMetabolism BiochemicalAssays

IntracellularpHplaysanimportantmodulatingroleinmanycellularevents,includingcellgrowth,calciumregulation,enzymaticactivity,receptor-mediatedsignaltransduction,iontransport,endocytosis,chemotaxis,celladhesionandothercellularprocesses.pH-sensitivefluorescentdyeshavebeenwidelyappliedtomonitorchangesinintracellularpHinrecentyears.ImagingtechniquesthatusefluorescentpHindicatorsalsoallowresearcherstoinvestigatetheseprocesseswithmuchgreaterspatialresolutionandsamplingdensitythatcanbeachievedusingothertechnologiessuchasmicroelectrode.Amongthem,2",7"-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein(BCECF)isthemostpopularpHprobesinceitcanbeusedtomonitorcellularpHratiometrically.However,allthecommercialBCECFAMisacomplexmixtureofatleastthreeisomerswithdifferentratiosfrombatchtobatch,complicatingtheBCECFapplications.BCFLisdevelopedtoovercomethisisomerdifficultyassociatedwithBCECFAM.AsBCECF,BCFLexhibitsapH-dependentdualexcitation,essentiallyidenticaltoBCECF.IthaspKaof~7.0,identicaltoBCECFtoo.AswithBCECF,thedualexcitationspectrumofBCFLwithanisosbesticpointat454nmshouldmakeBCFLagoodexcitation-ratiometriepHindicator.BCFLratiometricimagingmakesintracellularpHdeterminationessentiallyindependentofseveralvariablefactors,includingdyeconcentration,pathlength,cellularleakageandphotobleachingrate.BCFL,AMisasingleisomer,makingthepHmeasurementmuchmorereproducIBLethantheBCECF,AM,whichisconsistedofquiteafewdifferentisomers.

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.Preparecellsasdesired.Forexample,plateadherentcellsovernightingrowthmediumat40,000to80,000cells/well/100µLfor 

      96-wellor10,000to20,000cells/well/25µLfor384-wellplates.

Note: Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.

2.PrepareRatioWorks™BCFL,AMdye-loADIngsolution:

2.1   Preparea10to20mMstocksolutionofRatioWorks™BCFL,AMinhigh-quality,anhydrousDMSO.Thestocksolutionshouldbeusedpromptly.Anyunusedsolutionneedtobealiquotedandrefrozenat<-20^oC.

Note:Avoidrepeatedfreeze-thawcycles,andprotectfromlight.

2.2   Preparea5-50uMRatioWorks™BCFL,AMdye-loadingsolutioninHanksand20mMHepesbuffer(HHBS).

 

3. RunpHAssay

3.1   Add100µL/well(96-wellplate)or25µL/well(384-wellplate)RatioWorks™BCFL,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

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