3h arachidonic acid

Measurement of the rate of arachidonic acid incorporation into cellular phospholipid pools was accomplished by kinetic radiolabeling using [³H]arachidonic acid substrate. Briefly, one million cells of each cell line were seeded onto 6-well plates, and grown until they reached 80% confluence. The next day serum free medium was added and the cells were incubated with 0.5 μCi of [³H]arachidonic acid (PerkinElmer) briefly for either five, fifteen, or 30 min. Following pulse labeling, the cells were rinsed with phosphate buffer saline (PBS) twice, and then total lipid extracts were generated as previously described.^{68 (link)} Total lipid extracts were separated via two dimensional thin-layer chromatography (TLC) using chloroform/methanol/ammonium hydroxide/water (60:33.33:2.66:4, v/v) as the first dimension followed by chloroform/methanol/acetic acid/water (64:8:10:2, v/v) as the second-dimension solvent systems. Individual phospholipid species were identified by migration with respect to mass standards, spots resolving with the PI, PC, PE, PS, PG, or PA standard were scraped from the TLC plate, and the radioactivity was quantified with a liquid scintillation counter. [³H]-containing phospholipids were normalized to the amount of cellular protein, as quantified by the BCA assay (Pierce).