Quantifying Plasma Membrane GLUT4 in C2C12 Cells

C2C12 transfected with 5 µg of pHA-Glut4-GFP plasmid and 10 µg of either pCMV-DNAJB3 or pCMV were plated on glass-bottom dishes. After stimulation with 100 nM of Insulin (Sigma Aldrich, St. Louis, MO), they were fixed with 4% paraformaldehyde without permeabilization and subjected to HA staining using a rabbit monoclonal anti-HA antibody (Rockland, Limerick, PA) followed by Alexa Fluor 594-conjugated goat anti-rabbit IgG (Abcam, Cambridge, UK). The Alexa Fluor 595/GFP ratio was determined by quantitative fluorescence microscopy as described previously^{27 (link)}. To avoid cell selection bias fields, cells expressing the HA–Glut4-eGFP were randomly chosen in the GFP channel blinded to the expression of HA–Glut4-GFP on the plasma membrane (Alexa Fluor 594 channel). Images were collected in both GFP and Alexa Fluor 594 channels. To optimize the dynamic range of the assay, exposure times for the channels were independently set to maximize the signal while minimizing the number of cells with expression levels above saturation. Once set for each channel, all images in that channel were collected at the same exposure. The fluorescence intensities of GFP and Alexa 594 were quantified at the single-cell level. Mock- transfected cells were used in parallel to correct for fluorescence resulting from non-specific binding of the primary and/or secondary antibodies.