Dissociation of Gonadal Adipose Tissue

A single-cell suspension was obtained using the tissue dissociation methods described previously [30 (link),31 (link),32 (link),40 (link)], with slight modifications. The GA was minced with scissors in a 35 mm dish containing ~100 μL dissociation media consisting of Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher; Waltham, MA, USA), 600–700 U/mL Collagenase Type II (Worthington Biochemical; Lakewood, NJ, USA), and 0.5 U/mL Dispase (Thermo Fisher) for 3 min and placed on ice. Minced tissue was then incubated in 10 mL dissociation media at 37 °C with gentle agitation for 1 h. Enzymatic digestion was quenched via the addition of growth media (DMEM containing 20% HyClone characterized fetal bovine serum (FBS; GE Healthcare, Lot #A00168; Chicago, IL, USA) followed by serial filtration of the cell suspension through 100 µm and 70 µm cell strainers (Corning Life Sciences; Tewksbury, MA, USA).