Cell culture and induction of differentiation of 3T3-L1 pre-adipocytes

Extensive information has been obtained using cell culture models such as 3T3-L1 cells to study adipocyte differentiation [8, 24]. Culture and differentiation of 3T3-L1 preadipocytes were performed as described in previous studies [25–27]. Briefly, 3T3-L1 cells, kindly provided by Dr. Brasaemle of the Rutgers University, were seeded at 2 × 10⁵/well of 6-well plates and cultured in DMEM medium supplemented with 10 % FBS, 30 μM biotin, 20 mM of glutamine, 1U/ml of penicillin/streptomycin. After confluence was reached, cell differentiation was induced by culturing in DMEM containing 1 μM insulin, 1 μM dexamethasone, and 0.5 mM of 3-isobutyl-1-methylxanthine (IBMX). After 48 h, induction media was removed and replaced with DMEM containing 10 % FBS, 1 μM insulin, 20 mM of glutamine and 1U/ml of penicillin/streptomycin, and renewed every 2 days. Lipid droplets in differentiated adipocytes (day 6 to day 10) were identified under microscope and after Oil Red O staining. In subsequent experiments, MTP inhibitors (MTPi), kindly provided by Dr. David Gordon of Bristol-Myers Squibb, were added to the media after cell induction. Fresh doses of MTPi were renewed every 2 days (D0-D10).