Beckman optima tlx ultracentrifuge

The fluorophore calcein (from Sigma-Aldrich) was solubilized at a concentration of 100 mM with 50 mM KPi, and the pH was adjusted to 7.0 using aliquots of 4 M KOH. The stocked vesicles (2 mg of lipid) were pelleted by ultracentrifugation (280,000 × g, 4 °C, 20 min with a TLA 100.1 rotor in a Beckman Optima TLX Ultracentrifuge; Beckman Coulter Life Sciences, Indianapolis, IN) and resuspended in 0.9 mL of 89 mM KPi, pH 7.0. calcein was added to the liposome solution at a self-quenching concentration (10 mM) and enclosed in the vesicles by 3 cycles of rapid freezing in liquid nitrogen and thawing at 40 °C (or 60 °C for mixtures containing DPPC). Thus, the osmolality of the liposomutle lumen (filled with 10 mM calcein plus 89 mM KPi pH 7.0) is ~190 mOsmol/kg, which equals the osmolality of the assay buffer (100 mM KPi pH 7.0). After extrusion through a 200 nm polycarbonate filter at 20 °C (or 60 °C for mixtures containing DPPC) to homogenize the vesicles, they were eluted through a 22-cm-long Sephadex-G75 (Sigma-Aldrich) column pre-equilibrated with the assay buffer to remove the external calcein. The collected 1 mL fractions containing the calcein-filled vesicles were identified by eye using an ultraviolet lamp (for fluorophore excitation) and diluted in a total volume of 10 mL of the assay buffer.

Skeletal muscle samples were homogenized in Lysis Buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM Na₂H₂P₂O₇, 1 mM b-CH₃H₇O₆PNa₂, 1 mM Na₃VO₄, 1 mM PMSF, 1 mg/mL leupeptin, and 1% (v/v) Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) using an UltraTurrax homogenizer, and then centrifuged at 16,000× g in a Beckman Optima TLX Ultracentrifuge (Beckman Coulter S.p.A., Milan, Italy) for 15 min at 4 °C. The supernatants were ultra-centrifuged at 40,000× g in a Beckman Optima TLX Ultracentrifuge for 20 min at 4 °C. The protein concentrations of the supernatants were determined by the method described previously.

Tissue samples of vWAT were homogenized in Lysis Buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM Na2H2P2O7, 1 mM b-CH3H7O6PNa2, 1 mM Na3VO4, 1 mM PMSF, 1 mg/mL leupeptin, and 1% (v/v) Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) using an UltraTurrax homogenizer, and then centrifuged at 16,000× g in a Beckman Optima TLX Ultracentrifuge (Beckman Coulter S.p.A., Milan, Italy) for 15 min at 4°C. The supernatants were then ultracentrifuged at 40,000 x RPM in a Beckman Optima TLX Ultracentrifuge for 20 min at 4°C.The protein concentrations of the supernatants of the centrifuged lysates were determined using Bio Rad’s DC method (Bio Rad Laboratories, s.r.l., Segrate, Italy).