Metabolic testing of reimplanted exBAT in mice. (a) Illustration of experimental design for studies of ex vivo browning of epididymal WAT and allogeneic injection (the allogeneic implantation was designed to minimize stress and background weight loss of mice): (1) donor mouse is sacrificed via CO2 asphyxiation; (2) epididymal WAT fragments are harvested from sacrificed donor mouse via necropsy; (3) WAT fragments are minced by passaging through a 19 gauge needle; (4) WAT fragments are cultured for 3 weeks in media with and without browning factors; (5) fragments are removed from media and washed with PBS; (6) fragments are injected subcutaneously into age-matched, sex-matched recipient mouse. (b) H&E (top) and UCP1 IHC (bottom) staining of cultured tissues (10 days) prior to implantation (note: visceral adipose tissue). Scale bar: 50 µm. (c) Change in body mass 15–17 weeks post-implantation (see Supplementary Fig. 4 for entire weight-loss data). Drop in body weight is due to entry into metabolic chambers from week 15–16, and subsequently exposure to cold temperature from week 16–17. Error bars indicate SEM, n = 8 for each group. (d) Percent fat mass of total body mass for control and experimental groups. Percent fat mass is measured prior to entering the metabolic chambers (15 weeks post-implant, p = 0.056), after 1 week in the chambers at 25 °C (16 weeks post-implant), and after 1 more week in the chambers at 8 °C (17 weeks post-implant). Error bars indicate SEM, n = 8 for each group.