HEK293 cells (7.5 × 106) were grown in a 10 cm2 tissue culture dish and treated with media containing the FA precursors. Sodium salts of the FAs were conjugated with freshly made fraction V FA-free BSA (Sigma, St. Louis, MO) in a ratio of 4:1 FA: BSA for 20:5n3 (EPA U-99-A) (Nuchek, Elysian, MN), 24:0 (lignoceric acid, N-24-A) ( Nuchek, Elysian, MN), and the VLC-PUFA 34:5n3 (BASF Pharma, Florham Park, NJ). Cells were treated with 30 μg/ml of the fatty acid in media for 72 h. Following treatment, cells were harvested and washed once in 0.1 M PBS containing 50 μM of fraction V FA-free BSA (Sigma, St Louis, MO) to sequester excess free FA, followed by an additional wash with PBS only. The cell pellets were stored at −80 °C until further processing for lipid analysis.
Gyening Y.K., Chauhan N.K., Tytanic M., Ea V., Brush R.S, & Agbaga M.P. (2022). ELOVL4 Mutations That Cause Spinocerebellar Ataxia-34 Differentially Alter Very Long Chain Fatty Acid Biosynthesis. Journal of Lipid Research, 64(1), 100317.
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