Palmitate bsa

RAW264.7 (ATCC) macrophage cell line was maintained in RPMI1640 medium (Invitrogen) containing 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen) and 1% Pen/Strep (Invitrogen). 3T3-L1 preadipocytes, a preadipocyte cell line commonly employed as a preadipose cell model [17 (link)], were maintained in DMEM medium (Invitrogen) containing 10% bovine serum (Invitrogen), 1% Pen/Strep (Invitrogen), and 1mM sodium pyruvate (Invitrogen). Cells were incubated at 37°C in a humidified 5% CO₂/95% air atmosphere. Differentiation of 3T3-L1 preadipocytes into mature adipocytes was performed using insulin (Sigma), dexamethasone (Sigma), and 3-isobutyl-1-methly-xanthine (Sigma) for 8 days as described [18 (link)]. Macrophage M1 phenotype was induced by culturing RAW264.7 in presence of 20ng/mL of mouse recombinant IFN-γ (BD Pharmingen) for 12h followed by 100ng/mL LPS (Sigma) stimulation for another 12h. M2 activation of macrophages was induced by either 20ng/mL IL-4 (Biolegend) or 20ng/mL IL-13 (Biolegend) for 12h. Palmitate-BSA (Sigma) complex preparation was performed as described previously [19 (link)]. Briefly, palmitate was dissolved in 95% ethanol at 60°C and mixed with pre-warmed 10% FFA-free BSA (Sigma), yielding a stock concentration of 7.5mM.