1)Remove2500µlaliquotsofsupernatantintoscintillationvials,addscintillationfluidandcount.2)Aliquot1.6mlsoftheremainingsupernatantintoeachoftwopyrexglasstubes.3)Add6mlsofchloroform-methanol,1:2,andvortexvigorously.-->Stillhaveamonophaseanditmaybebesttoallowthismonophasetoequilibrateforalittlewhile.4)Removealllargedebrisbyspinningat3,000rpmfor5mins.5)Transferthesupernatanttoanewpyrexglasstubeandvortexagain.Donottransferanyofthepellet,evenifitisnecessarytosacrificesomeofthesample.6)Add2mlofchloroformtobreakphasesandvortexvigorously.7)Add2mlofwaterandagainvortexvigorously.8)Spinsamplesat3,000rpmfor5mins.tocompletelyseparatephases.9)Aspirateoffthetopphaseandtheinterface.-->Removeenoughsuchthatnoaqueousmaterialremainsattheminiscus.10)Thelowerphaseshouldconstitute~4ml.Transfer3.5-3.7mlofthistoanewpyrexglasstube.-->Thisisagoodpointtostopbystoringthetransferredsamplesinthe-200Cfreezer.11)DrydownsamplescompletelyandresUSPendlipidsin60µlofchloroform.12)Use20µltospotalinearKTLCplateforTLCsystem#1,use20µltospotalinearKTLCplateforTLCsystem#2.

TLC:

A)AllowTLCtankstoequilibrate1-2hrswithsolventpriortorunning.SYSTEM#1-->SeparatesPA,TXB2,HHT,HETE&AA.ethylacetate/2,2,4-trimethylpentane/aceticacid/H2Oataratioof90:50:20:100SYSTEM#2-->SeparatesDAG&AA.benzene/diethylether/ethanol/NH3ataratioof100:80:4:0.2B)FollowingTLCremoveplatesandallowtoevaporatecompletely.Thisshouldtakeafewhours.-->Ifanotherplateneedstoberuninthesametankthenallowtanktoequilibrateanotherhourfollowingremovalofthefirstplate.C)Sprayplatewithenhance,spotwithrADIoactiveink,wrapinsaranwrapandplaceinacassette(nointensifyingscreennecessary).-->Spotinkinlocationswhereitwillnotbedisturbed.Inotherwords,donotputspotsbelowthesampleapplicationlevels,sincetheplatewillbeheldupsidedowntoscrapespots.18)Exposefilmforaminimumof40hours(3daysismuchbetter)inthe-800Cfreezer.19)Allowenhancetocompletelyevaporate.-->Sincetheplateiswrappedinsaranwrapduringdeveloping,enhanceremainswetontheplate,andthismustbeevaporatedinordertoretainallofthesilicawhenscraping.20)Followingfilmdevelopment,markofflocationsofspots.21)Holdplateupsidedownandtiltedoverglassineweighpapersandscrapespots.-->Addscrapingsintoprefilledplasticscintillationvialstominimizelossofsilicaandsample.22)Vortexvialsandcountspots,countvialsagain24hourslater.