humidified atmosphere containing 5 co 2 in air 3 h anandamide 0 2 u03bcci of 221 ci mmol of 3 h anandamide was added and the incubation continued for 1 hour control cells contained no inhibitor at the end of the incubation the cells were washed once with cell culture media and removed from the plates after a brief incubation with 2 ml of 0 05 trypsin in 0 53 mm edta solution at 37 u00b0 c the amounts of 3 h anandamide 3 h phospholipids and 3 h arachidonate in the cells and media were quantified by liquid scintillation counting of the silica scraped from the appropriate areas of the plate after quenching the reaction with chloroform methanol 1 1 extraction of the sample from the organic phase and tlc analysis on channeled silica gel coated plates with a solvent system consisting of the organic layer of an ethyl acetate hexane acetic acid water 100 50 20 100 mixture the level of 3 h anandamide found in the neuroblastoma cells incubated with palmitylfsulfonyl fluoride with phenylmethylsulfonyl fluoride and in control cells is shown in fig1 nanomolar amounts of palmitylsulfonyl fluoride were sufficient so that over 50 of the radioactivity was found in anandamide rather than in anandamide cleavage products such as arachidonate this result indicates that palmitylfsulfonyl fluoride is highly effective at inhibiting anandamide amidase concentrations greater than 10 micromolar of phenylmethylsulfonyl fluoride were required to achieve comparable levels.