Rate of Total Fatty Acid Synthesis From [1-14C]acetate

After 24 h incubations of U87MG cells with C8 or C10, the rate of total fatty acid and cholesterol synthesis was followed by adding [1-¹⁴C] acetate (1.56 mCi/mmol) to cells. The reaction was stopped, after 1 h, with 1 ml of 0.5 N NaOH. Cells were scraped off the plates and transferred to test tubes, and after ethanol addition, samples were subjected to saponification. At the end of the saponification reaction, cholesterol was extracted three times with 4 ml of petroleum ether. The suspension was then acidified with 0.5 ml 7 N HCl and fatty acids were extracted three times with 4 ml of petroleum ether. After solvent evaporation, the radioactivity associated with labeled cholesterol and fatty acids was measured (Vergara et al., 2017; Giudetti et al., 2019).