CholineLabelingandTNFTreatmentofHL-60CellsContributor:SupryaJayadevDate:Aug.6,19911)Growcellstoadensityof5-8X105cells/mlinRPMI1640containingserum.2)Pelletcellsandwash1timewithroomtemperaturePBS.3)ResUSPendfinalpelletinanappropriatevolumeofprewarmedserumfreemediaandsupplementwithITS(4ml/Lshouldyield5mg/Linsulinand5mg/Ltransferrin).-->"Appropriate"=bringcellstoafinalconcentrationof2X105cells/ml.4)Add(0.5µl/mlmedia)[3H]cholinechloridetoget~0.5µCi/ml.-->Specificactivityofthe[3H]cholinechlorideisusuallyaround80Ci/mmol.5)Incubatecellswithlabelforaperiodof48or72hours.-->Ifexperimentrequiresmorecellsthenincubatelongerorelseincubateonlyfor48hrs.Beconsistentinthelabelingperiodsincecellcyclechangescanhaveaneffectonresults.6)Washoutlabelbypelletingcellsandverygentlywashing1timewithroomtemperaturePBS.7)Followingwash,resuspendthepelletin1/2to1/3theoriginalvolumeusingprewarmedserumfreemediaandcountcells.8)SeedcellsintoT-25flasksfortreatment:10mls/flask.-->Thus,have4.5mlduplicatesfromeachflask.9)Allowcellsto"rest"inincubatorfor2-4hrs(Inapinch,canleaveovernight).Alwayskeeptrackofresttime!!!-->Equilibrateforlongerperiodstopreventcontrolsfromfluctuating.10)Treatcellswith30nMTNFinBSAforappropriateperiod.

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