Glut4 h61

The cells were washed with PBS, trypsinized, resuspended in PBS, and centrifuged two times for 7 min at 400 × g at room temperature. This was followed by a 60 min incubation at 4°C in blocking solution (2% FBS, 2% bovine serum albumin, 0.2% fish gelatine, in PBS). Finally, the cells were resuspended in cold PBS containing 5% FBS. Aliquots of 3 × 10⁵ cells/tube were incubated for 30 min at 4°C with monoclonal mouse antibodies reactive with the insulin receptor β subunit (1:300, BD Biosciences), with GLUT1 (1:100, Invitrogen), with rabbit polyclonal antibody reactive with GLUT2 (H-67), and GLUT4 (H-61), both at 1:100, (Santa Cruz Biotechnology Inc). The antigen-bound antibodies were visualized by a 30 min incubation at 4°C with Alexa Fluor488-conjugated donkey anti-rabbit and Atto550-conjugated donkey anti-mouse IgG, both 1:100 (Sigma-Aldrich, Poland). Stained cells were washed with 1 ml PBS, resuspended in 300 μl of PBS containing 5% FBS, and analyzed by flow cytometry (FACScan Flow Cytometer, BD, USA). Background fluorescence, assessed with IgG isotype controls, was subtracted from the corresponding samples during analysis.