2 deoxy d glucose

Mouse SVFs and human SGBS preadipocytes were grown and differentiated in 24-well plates, and starved in serum-free and glucose-free medium for 3 h prior to the assay. After washing 2 times with pre-warmed KRH buffer [50 mM HEPES, 137 mM NaCl, 4.7 mM kCl, 1.85 mM CaCl₂, 1.3 mM MgSO₄, pH = 7.4], cells were treated with or without insulin (100 nM) in KRH buffer for 30 min, followed by the addition of 1 μCi ml⁻¹ [³H] 2-Deoxy-D-Glucose (PerkinElmer) and 100 μM 2-Deoxy-d-Glucose (Cayman) for 15 min. Reactions were terminated by washing the cells with ice-cold KRH buffer for three times. Cells were then lysed by M-PER™ Mammalian Protein Extraction Reagent (Thermo Scientific), and the incorporated radioactivity was determined by liquid scintillation counting. A small aliquot of each cell lysate was used for protein quantification and normalization of radioactivity counting. For ex vivo glucose uptake on adipose tissue explants, ~20 mg tissues were weighed and cultured in one of the 24-well. After following the same procedure as described above, tissues were digested with 0.5 M NaOH overnight. Radioactivity measurements on the tissue lysates were normalized against the tissue weight per well.