Fatty Acid Conjugation to BSA

Fatty acids are fat-soluble molecules and therefore insoluble in the cell culture media in their native form; furthermore, free fatty acids travel in the bloodstream bound to albumin. Thus, to allow for fatty acid solubilisation in the cell culture media and mimic fatty acid physiological circulating conditions, both PA and LA were conjugated to BSA as described previously (15). Low-endotoxin BSA was used to prevent potential lipopolysaccharide contamination as reported previously (39). Fatty acids were dissolved in 0.1 M NaOH in a water bath at 70°C to yield a final concentration of 20 mM. BSA was solubilised in serum and penicillin-streptomycin–free DMEM at 55°C and fatty acid mixed with the fatty acid solutions to obtain a 1:4 molar ratio (fatty acids 2 mM: BSA 0.5 mM). The fatty acid–BSA mix was vortexed and then incubated for 10 min at 55°C and cooled to room temperature before being filter sterilised. Conjugated fatty acids were stored at −20°C before use.