Arachidonic GAP 1-13
release. Cells were cultured on poly-l-ornithine-coated 24-well plates (Nunc) to 50% confluence. 0.1 μCi [3H]-AA ([5,6,8,9,11,12,14,15-3H]-arachidonic acid, New England Nuclear Corp. GesmbH, Wien, Austria) was added in each well and the cells cultured for another 20 h. The incubation medium was removed and the cells were washed twice with the culture medium without serum but supplemented with 2 mg/ml bovine serum albumin (BSA). The stimulations with Ox-A were performed at 37 °C in 250 μl/well of this same medium. After 7 min stimulation, 200 μl of the medium from each well was transferred to an Eppendorf tube on ice. These samples were spun down for 1 min at 4 °C and 150 μl of the medium was transferred to a radioactive decay
scintillation tube and the scintillation cocktail (HiSafe 3, Wallac-PerkinElmer, Turku, Finland) was added. Cell remnants on the 24 well plates were dissolved in 0.1 M NaOH and the scintillation cocktail was added. The radioactivity was counted in a scintillation counter after allowing the samples to set for 24 h.