Overview

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Ex/Em(nm)	490/525
MW	N/A
CAS#	N/A
Solvent	N/A
Storage	F/D/L
Category	IonChannels
Related

Potassium(K+)ionchannelplaysanimportantroleinregulatingfundamentalBIOLOGicalprocessesincludingheartrate,hormoneandneurotransmittersecretion,waterandelectrolytebalance.Potassiumionchannelhasbeenconsideredasdrugtargetsfordiseaseindicationsincludingarrhythmia,pain,diabetes,neurologicaldysfunctionsetc.ThepermeABIlityofTl+throughK+channelhasbeenwidelyusedtoassayK+channel.ThecellsthatexpressK+channelofinterests(e.g.hERG,Kv1.3,Kir2.1,KATP)arepre-loadedwithaTl+sensitivedye.Thedyeisnon-fluorescentandispermeabletocellmembrane.Onceinsidethecell,thenon-fluorescentAMesterdyeiscleavedbyendogenousesteraseintoanegativelychargesdyethatstaysinsidecells.WhenastimulusbuffercontaininglowdoseofTl+isaddedtocells,theTl+flowsacrosstheK+channelandbindstoTl+sensitivedye,generatingafluorescentsignal.ThissignalisproportionaltotheactivityofK+channel.IfanantagoNISTorantagonistisaddedtothecells,thefluorescentsignaldecreasesorincreasesrespectively,toreflecttheinhibitedorstimulatedactivityofK+channel.

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1. Preparecells:
   1. Foradherentcells:Platecellsovernightingrowthmediumat40,000to80,000cells/well/100µLfora96-wellpolyD-lysineplateor10,000to20,000cells/well/25µLfora384-wellpoly-D-lysineplate.
   2. Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletinequalamountofHHBSandTl-520AMdye-loADIngsolution(seeStep2.3below)at125,000to250,000cells/well/100µLfora96-wellpoly-D-lysineplateor30,000to60,000cells/well/25µLfora384-wellpoly-D-lysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiments.
      Note1:Eachcelllineshouldbeevaluatedontheindividualbasistodeterminetheoptimalcelldensityfortheintracellularthalliummobilization.
      Note2:ForHEK293-KCNH2cellsthatweusedinthisassay,werecommendtoplatecellsat20,000cells/well/100µLina96-wellpoly-Dlysineplateforovernight.Thecellsshouldreach80-90%confluencyontheexperimentday.


2. Preparedye-loadingsolution:
   1. Make500XTl-520AMstocksolution:Add20µL(forCat#36550)or200µL(forCat#36551and36552)ofDMSOintothevialofTl-520AM(ComponentA),andmixwell.
      Note:TheunusedTl-520AMstocksolutioncanbestoredat-20°Cinasingle-usealiquots.
   2. Make1Xassaybuffer:Add1mlof10XPluronic®F127Plus(ComponentB)to9mlofHHBS(ComponentC).
   3. Makedye-loadingsolution:Add20µLofdyestocksolution(fromStep2.2)into10mlof1Xassaybuffer(fromStep2.3),andmixwell.


3. Incubatecellswithdye-loadingsolution:
   1. Addequalvolume(suchas100µL/wellfora96-wellplateor25µL/wellfora384-wellplate)ofDye-LoadingSolution(fromStep2.3)toyourcellplatewithoutremovingthecellmedium.
   2. Incubatethedye-ladingplateinthecellincubatorfor1hour.


4. Prepare5Xstimulussolution:
   1. Make1Xchloridefreebuffer(ComponentD):Dilute5Xchloridefreebufferto1XchloridefreebufferinddH2O.
   2. MakeTl2SO4solution(Notprovided,SigmaCat#208191):DissolveTl2SO4inultrapureH2Otofinalconcentrationof80mM.
      Note:Tl2SO4istoxic.Takenecessaryprecautionstopreventinhalationandskincontact
   3. Preparea5XStimulussolutionbydilutingligands(fornon-voltagegatedpotassiumchannels)orK2SO4(forvoltagegatedpotassiumchannels)withTl2SO4in1XChloride-freebuffer.
      
      Pleaseusethefollowingtableasareference(forvoltagegatedhERGchannelinHEK293-KCNH2cells),concentrationofTl2SO4andK2SO4(forvoltagegatedpotassiumchannels)orligands(fornon-voltagegatedpotassiumchannels)usedfortheassayshouldbeoptimizedforeachtargetchannelandcelltype.
      
      

      Component	Volume	5XConcentration	FinalConcentration
      5XChloride-freeAssayBuffer(ComponentD)	2mL
      250mMPotassiumSulfate(K2SO4)(ComponentE)	1mL	12.5mM	2.5mM
      80mMThalliumSulfate(Tl2SO4)(notprovided)	0.5mL	2.5mM	0.5mM
      ddH2O	5.5mL
      Total:	10mL



5. Runthepotassiumassay:
   1. PreparetheK+channelantagonistsoragonist(from4.3)inHHBS.
   2. Incubatetheplatewithantagonists(forinhibitorystudy)inthecellincubatorfor30minutes.
   3. Add50µL/well(fora96-wellplate)or12.5µL/well(fora384-wellplate)of5XstimulusbufferwithFLIPR,FDSSorFlexstation.RuntheexperimentwithafiltersetofEx/Em=490/525nm.Readtheplateevery1–2secondsfor3minutes.

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