2.6. Conjugation of Palmitate to Bovine Serum Albumine (BSA)

For conjugation of palmitate (Sigma) with BSA (Sigma), 538.4 mg ultra-low fatty acid BSA was diluted in 12 mL autoclaved, 37 °C warm 150 mM NaCl, followed by incubation at 37 °C till BSA is dissolved. Then, 9.18 mg palmitate was diluted in 6.6 mL of warm 150 mM NaCl and then heated at 70 °C with stirring till the solution was clear. Thereafter, 8.25 mL warm BSA solution was transferred to a new tube and palmitate solution was added slowly to BSA solution. The conjugate of palmitate to BSA was stirred for 1 h at 37 °C. Then, 1.65 mL 150 mM NaCl solution was added to the conjugate and pH was adjusted to 7.4 with 1 M NaOH. The conjugate solution was aliquoted and stored at −20 °C. BSA vehicle was prepared by mixing 3 mL BSA solution with 3 mL 150 mM NaCl. BSA vehicle was aliquoted and stored at −20 °C. The molar ratio of conjugated palmitate:BSA was as 6:1. Prior cell treatment with palmitate:BSA conjugate, both BSA and palmitate were thawed at 37 °C for 10 min.