Isolation of clones that stably express GFP and luciferase. B16-F10 cells were transduced with the GFP-P2A-luc lentiviral vector and GFP- positive cells were purified by FACS (> 95%). (A) Single cell clones were isolated by limiting dilution, expanded in vitro and tested to confirm stable expression of GFP. The specificity and sensitivity of luciferase expression was demonstrated by serial dilution of the B16-F10 clones and appropriate controls (negative = untransduced, or cells transduced with GFP only). BLI images were taken prior to or following the addition of D-luciferin substrate (B) using a Xenogen IVIS-100. (C) Complete separation of GFP and luciferase protein was confirmed by SDS-PAGE and immunoblotting using anti-GFP (Cat. # 632380, BD Bioscience) and anti-P2A antibodies . Separated GFP protein migrated at 27 kDa, whereas the GFP-P2A product will migrate at a higher molecular weight (~29 kDa). Un-separated GFP-P2A-luc protein was predicted to migrate at 90 kDa and was not present. Actin was used as a loading control and detected also by immunoblotting (Cat. # A2103, Sigma).