| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 630/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | IonChannels ChlorideChannels |
| Related | pHandIonIndicators BiochemicalAssays |
1.Preparecells:
1.1 Foradherentcells:Platecellsovernightingrowthmediumat40,000to80,000cells/well/100µLfora96-wellplateor10,000to20,000cells/well/25µLfora384-wellplate.
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletinpre-warmedassaybufferat125,000to250,000cells/well/100µLfora96-wellpoly-Dlysineplateor30,000to60,000cells/well/25µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiments
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
2.Prepareiodideassayreagents:
2.1 Warmallthereagentstoroomtemperaturebeforeuse.
2.2 Make1XI-sensorenhancersolution:Add50µLof100XIodidesensorenhancer(ComponentB)to5mLofsterileH2O,andmixthemwell.
Note1:1XI-sensorenhancersolutionisnotstable,usewithin2hoursafterthedilution.
Note2:EachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimaldilutionofI-sensorenhancersolution.Wenoticedthat0.1XI-sensorenhancersolutionworksevenbetterforsomecelllines.
2.3 Make1Xcelllysisbuffer:Addthewholevialof10XCellLysisBuffer(ComponentD)to45mLofsterileH2O,andmixthemwell.
Note:5mLof1Xcelllysisbufferisenoughforoneplate.Storeunused1Xcelllysisbufferat4oC.
3.Foriodideeffluxassay:
3.1 Aspiratethegrowthmediumfromthecellplate.
3.2 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofpre-warmedI-LoADIngBuffer(ComponentC)andincubatefor2-4hours.
3.3 Aspiratetheiodideloadingbuffercompletely,andwashthecellswithDPBSorHBSSatleast3times.
3.4 TreatthecellswithagoNISTinHBSSbufferfor5minutes.
Note:Forantagonistsscreen,incubatethecompoundswithI-loadingbufferforatleastanadditional30minbeforethecellswerewashedwithDPBSorHBSSbuffer.
3.5 Aspiratethesupernatant.
3.6 Lysethecellsbyadding50µL/well(96-wellplate),or25µL/well(384-wellplate)of1Xcelllysisbuffer(fromStep2.3).
3.7 Performtheiodideassay(SeeStep5).
4.Foriodideinfluxassay:
4.1 Aspiratethegrowthmediumfromthecellplate.
4.2 Add100µL/well(96-wellplate),or25µL/well(384-wellplate)ofpre-warmedI-LoadingBuffer(ComponentC)withtestcompounds,andincubatefor5minutes.
4.3 Aspiratetheiodideloadingbuffercompletely,andwashthecellswithHBSS3times.
4.4 Lysethecellsbyadding50µL/well(96-wellplate),or25µL/well(384-wellplate)of1Xcelllysisbuffer(fromStep2.3)
4.5 Performtheiodideassay(SeeStep5).
5.Runiodideassay:
5.1 Add50µL/well(96-wellplate),or25µL/well(384-wellplate)ofIodideBlue™sensor(ComponentA)tothewellsthatcontaindifferentconcentrationsofpotassiumiodide(fromStep3.7orStep4.5).
5.2 Add50µL/well(96-wellplate),or25µL/well(384-wellplate)of1Xiodidesensorenhancersolution(fromStep2.2)intothemixture(Step5.1).
Note:Forsomecelllines,youmightneedtodiluteenhancersolutiondownto0.1X.
5.3 Incubateatroomtemperaturefor10sec-10min.
Note1:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalincubationtime.
Note2:Thebluecolormaychangetoyellowwithinsecondstominutesduetothepresenceofahighconcentrationofiodide.
5.4 Monitorabsorbanceat630,380,or405nm.
| References&Citations |  PrinterFriendlyVersion |
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