In vitro binding specificity of IRDye® 800CW EGF for tumor cells in culture.A431 cells cultured to 90% confluence in 96-well plates were incubated with increasing concentrations of IRDye® 800CW EGF (diamonds) or unconjugated, non-reactive IRDye®800CW (circles) for 2 min, then washed, fixed, permeabilized and stained with TO-PRO-3 (A). For competitions, cells were incubated with a fixed concentration of IRDye® 800CW EGF (70 nM) containing the indicated concentrations of unlabeled EGF (B), or C225, an EGF receptor blocking antibody (C). All plates were scanned at two wavelengths on the Aerius® near infrared scanner. The 800 nm signal, normalized to the 700 nm control, is plotted as the mean ± SD of three replicate wells.