Adipogenic Differentiation Protocol

2d after reaching confluence, cells were differentiated in a serum-free complete differentiation media (DMEM/F12 with 0.5 mM IBMX, 100 nM insulin, 100 nM dexamethasone, 2 nM T₃, 10 μg/ml transferrin, 1 μM Rosiglitazone, 33 μM biotin and 17 μM pantothenic acid) (9 (link), 10 (link)). After induction in the complete differentiation media (3, 7 or 11 days), cells were maintained in DMEM/F12 with insulin (10 nM) and dexamethasone (10 nM) until harvest or use for metabolic experiments (d14 to d17). There was no evidence of cell toxicity due to the longer induction protocol as judged by the release of the intracellular enzyme LDH (data not shown). For testing the effects of FBS on adipogenesis, 0, 1, 3, 5 or 10% FBS was added to differentiation and maintenance media.