Propranolol

Stromal vascular (SV) fraction was obtained from subcutaneous adipose depots of two-month old mice as described.8 (link),20 (link),21 (link) Isolated SV cells were cultured in DMEM supplemented with 10% FBS, 100 units/mL penicillin, 100 mg/mL streptomycin and 25 ng/mL Amphotericin B (Sigma). White adipogenesis was induced as described.8 (link),21 (link) Beige adipogenesis was induced similarly, except for an addition of 5 nM indomethacin (Sigma) and 2 nM T3 (Sigma).20 (link) Fulvestrant (1 µM, Sigma), Estradiol (1 µM, Sigma), CL-316,243 (Tocris Bioscience), SR59230A (1 µM, Sigma), Propranolol (1 µM, Cayman Chemical, Ann Arbor, MI) or vehicles (ethanol, H₂O or DMSO in equivalent dilutions) were added to confluent cells with each media change. To induce thermogenic genes, cells were treated with 10 µM Forskolin (Sigma), 1 µM Norepinephrine (dissolved in H₂O with 0.125 mM ascorbic acid, Sigma) or 1 µM CL-316,243–8 hr for RNA isolation or 24 hr for immunostaining. “Sub-optimal beige media” - IBMX and Dexamethasone (Sigma) were removed. Oil Red O staining, Nile Red staining and immunostaining were done as described.8 (link),21 (link) Relative changes in cAMP levels were measured using cAMP-Glo™ Max Assay (Promega, Madison, WI) following induction in PBS and IBMX (500 µM, Sigma) with or without 10 µM Forskolin for 15 min.