Its premix insulin transferrin selenium

Adipose tissue SVF was isolated as described above. Cells were plated onto collagen-coated dishes and incubated at 10% CO₂. Gonadal SVF was maintained in growth media (60% pH7–7.4 low glucose DMEM, 40% pH 7.25 MCDB201 (Sigma M6770)), supplemented with 2% FBS (Fisher Scientific 03-600-511 Lot FB-002) 1% ITS premix (Insulin-Transferrin-Selenium) (BD Bioscience 354352), 0.1 mM L-ascorbic acid-2-2phosphate (Sigma A8960-5G), 10 ng/mL FGF basic (R&D systems 3139-FB-025/CF), Pen/Strep, and gentamicin. Inguinal SVF was maintained in DMEM/F12 (Invitrogen) supplemented with Glutamax, 10% FBS, Pen/Strep, and gentamicin. Upon reaching confluence, cultures were incubated with the adipogenesis induction cocktail (growth media supplemented with 5 mg/ml insulin, 1 μM dexamethasone, 0.5 mM isobutylmethyxanthine, and 1 μM rosiglitazone) for 48 hr. After 48 hr, the cells were maintained in growth media supplemented with 5 mg/ml insulin and 1 μM rosiglitazone until harvest.

For all cellular assays, 4–6 weeks-old C57BL/6 mice were utilized. PDGFRβ+ subpopulations were isolated from gonadal WAT SVF as described above. For spontaneous adipogenesis assays, sorted cells were plated in 48-well plates at a density of 4 × 10⁴ cells/well in growth media containing 2% FBS and ITS supplement [60% pH7–7.4 low glucose DMEM, 40% pH 7.25 MCDB201 (Sigma M6770), 1% ITS premix (Insulin-Transferrin-Selenium) (BD Bioscience 354352), 0.1 mM L-ascorbic acid-2-2phosphate (Sigma A8960-5G), 10 ng/mL FGF basic (R and D Systems 3139-FB-025/CF), Pen/Strep, and gentamicin] and incubated at 37°C in 10% CO₂. Media was replaced every other day. For induced adipogenesis, confluent cultures of FIPs were treated with an adipogenesis induction cocktail (growth media supplemented with 5 mg/ml insulin, 1 μM dexamethasone, 0.5 mM isobutylmethyxanthine, ±1 μM rosiglitazone) for 48 hr. After 48 hr., the cells were maintained in growth media.