(Technically, "Measurement of the Release of Arachidonic Acid plus Oxygenated Metabolites" would be more correct, but never mind).

Theoretically, this procedure is simple and straightforward. Yet it sometimes does not work. If that happens, do not blame the protocol below but rather the cells you are using, especially if they are tumor cell lines. When conducted as described below, arachidonic acid release experiments work wonderfully well in cells such as macrophages or mast cells.

Cells (usually, one million per ml) are labeled with 0.5 mCi/ml [3H]arachidonic acid (Amersham or NEN; sp. act. ~50-100 Ci/mmol) overnight in serum-containing medium.

Wash the cells four times with phosphate-buffered saline (containing 0.5 mg/ml bovine serum albumin), to remove the unesterified labeled fatty acid.

Leave the cells in serum-free medium (containing 0.5 mg/ml bovine serum albumin - this is very important!) for at least 1 h.

Add the stimulus for the time indicated. If inhibitors are to be used, it is recommended to add them 15-30 min before the stimulus. As a positive control use a phorbol ester/ionophore A23187 mix (at 100 ng/ml and 1-10 µM, respectively).

After the appropriate stimulation times, the reactions are stopped by placing the tubes/dishes/etc on ice. If cells in suspension are used, centrifuge at 1200 rpm for 5 min at 4°C to precipitate the cells. If adherent cells are used, carefully remove the incubation medium but centrifuge nevertheless, so as to spin down any dislodged cell.

An aliquot of the supernatant is mixed with scintillation cocktail and counted on a liquid scintillation counter.