F6428 40ml

Lipolysis measurements in WAT adipocytes obtained by abdominal biopsies were performed according to a standard protocol [31 (link), 41 (link)]. In brief, isolated fat cells / fatpad were incubated for 2 h at 37 °C in Krebs–Ringer phosphate buffer supplemented with glucose (8.6 µmol/ml), ascorbic acid (0.1 mg/ml) and BSA (20 mg/ml). Adipocytes were incubated in the absence (basal) or presence of increasing concentrations of noradrenaline or isoprenaline (stimulated). Basal and stimulated (noradrenaline and isoprenaline) lipolysis was measured as glycerol release and normalized per gram of fat tissue.
In addition, in vitro differentiated adipocytes were incubated with T-cell-derived CM, as described above. After 24 h media was collected from adipocytes and glycerol release was measured. Glycerol was quantified by the commercially available free glycerol reagent kit (F6428-40ml, Sigma-Aldrich) and Amplex ultra-red reagent (A36006, Invitrogen) as described previously [42 (link)]. Glycerol concentration was normalized to the protein amount in corresponding well measured by commercially available BCA kit (23,225, Thermofisher Scientific) [42 (link)].

ADSCs were seeded at 5 × 10³ cells per cm² in 12-well plates, grown for 5 days and then induced to differentiate into beige adipocytes for 21 days. Prior to the assay, cells were washed in DPBS twice and then in DMEM supplemented with 2% fatty-acid free BSA (Santa Crus, sc-500949) and Triascin C (5 μM). Cells were treated with FSK (20 μM) for the indicated time periods and supernatant samples were collected. Lipolysis assays were performed with free glycerol reagent (Sigma, F6428-40ML), according to the manufacturer’s instructions.