Cell culture and conditioned medium preparation of 3T3-L1 adipocytes

SVEC4-10 endothelial and 3T3-L1 preadipocytes were maintained in DMEM supplemented with either 10% FBS or 10% newborn calf serum (NBCS), respectively, and incubated at 37 °C in a humidified 5% CO₂ atmosphere. For adipocyte differentiation, 3T3-L1 preadipocytes were exposed to a differentiation medium (MDI) consisting of DMEM containing 10% FBS, 0.5 mM IBMX, 1.72 nM insulin, 1 µM dexamethasone, and 100 µM indomethacin for 48 h. Afterwards, stimulated cells were maintained in medium supplemented with 10% FBS and 1.72 nM insulin for an additional 6 days by replacing half of the medium every 2 days. Eight days after induction of differentiation, the culture medium of the mature 3T3-L1 adipocytes was collected and centrifuged at 500 xg for 10 min at 4 °C to remove cellular debris. The resulting supernatant was used for experiments requiring adipocyte-conditioned medium (Ad-CM).