Fatty acid free bsa

About 40 mg white adipose tissue from mice was cut into small pieces, and incubated at 37°C on a shaker at 80 rpm for 1 h in 1.5 mL Hank’s solution (140 mM NaCl, 5 mM KCl, 0.3 mM Na₂HPO₄, 0.4 mM KH₂PO₄, 4 mM NaHCO₃, 1 mM CaCl₂, 0.4 mM MgSO₄, 0.5 mM MgCl₂, 6 mM glucose) supplemented with 0.4 mg/mL collagenase (Sigma), 2% fatty acid free BSA (Equitech-Bio) and 0.1% glucose. Then the samples were filtered with 100 μm cell strainer (Corning), and centrifuged at 23×g for 2 min. The floating layer containing the adipocytes was transferred into 10 mL Hank’s buffer, and centrifuged at 48×g for 2 min. The floating layer was transferred into a new tube containing 300 μL Hank’s solution supplemented with 1mg/mL BODIPY 500/510 C1, C12 (Invitrogen) and 1% fatty acid-free BSA, briefly mixed, and incubated at 37°C for 5 min. The samples were filtered with 40 μm cell strainer (Corning) to keep the adipocytes on the mesh, and then the adipocytes were lysed with 200 μL 0.3 M NaOH to detect the fluorescence intensity of BODIPY 500/510 C1, C12.