O phenylenediamine dihydrochloride reagent

Cell surface GLUT4myc levels were assessed by an antibody-coupled colorimetric assay.[29 (link)] Briefly, L6-GLUT4myc myoblasts were cultured in 24-well plates until confluence and serum-starved for 4 h before incubation with either quercetin (50 μM) or vehicle (DMSO, 0.1%) for 18 h; insulin (100 nM) incubated for the last 15 min served as a positive control. At the end of incubation, cells were quickly washed with ice-cold PBS and put in the presence of an anti-c-myc antibody (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 60 min at 4°C. Cells were then washed and fixed in 3% paraformaldehyde for 3 min on ice. The fixative was neutralized by incubation in 10 mM glycine in ice-cold PBS for 10 min. Goat serum (5%) was used to block cells for 30 min before incubation for an additional 60 min with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG at 4°C (1:1,000 dilution; Cell Signaling Technologies, Danvers, MA, USA). Cells were then washed five times with ice-cold PBS and incubated with O-phenylenediamine dihydrochloride (OPD) reagent (1 ml/well) (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 30 min. Addition of 0.25 ml of 3 M HCl to each well stopped the reaction. The supernatant was collected and its absorbance was measured at 492 nm. Absorbance associated with nonspecific binding (primary antibody omitted) was used as a blank.