Contributor:SupryaJayadevDate:November4,19911)Growcellstoadensityof5-8X105cells/mlinRPMI1640containingserum.2)Pelletcellsandwash1timewithroomtemperaturePBS.3)ResUSPendfinalpelletinanappropriatevolumeofprewarmedserumfreemediaandsupplementwithITS(4ml/Lshouldyield5mg/Linsulinand5mg/Ltransferrin).-->"Appropriate"=bringcellstoafinalconcentrationof5X105cells/ml.4)Add[3H]arachidonicacidtoget~2µCi/ml(2X106µCi/ml???).-->MakesurefinalAAconcentrationisinthenMrange.5)Incubatecellswithlabelforaperiodof60minutes(2hours???).6)Removelabelbywashingcells3Xwithicecoldmedia.7)Aliquotwashedcellstoget5X105cells/mlagain.8)Treatwithappropriatecompound(andvehicle)forappropriatelengthoftime.9)Transfer4.5mlintoeachoftwoduplicateglasspyrextubesandmaintainonice.10)Spindowncellsinthetabletopcentrifugeat1,200rpm,40C,for5minutes.
