Lentiviral vectors designed to achieve equimolar expression of GFP and luciferase. (A) The third-generation lentiviral expression vector pHIV1SDm  containing 5' and 3' LTRs, long terminal repeat; RRE, rev response element; cPPT, central polypurine tract; CMV, cytomegalovirus promoter; GFP, enhanced green fluorescent protein; P2A, porcine teschovirus-1 2A motif; Wpre, woodchuck hepatitis post-transcriptional regulatory element; and self-inactivating (SIN) 3'LTR. The vectors contained (i) GFP alone as a control, or (ii) a GFP and firefly luciferase cassette (GFP-P2A-luc). (B) MCF-7 or (C) B16-F10 cells were transduced with a lentiviral vector containing the GFP-P2A-luc cassette and purified into 7 or 8 different populations based on increasing GFP expression by FACS. B16-F10 (20, 000) or MCF-7 (10, 000) cells/well were deposited in a black 96-well plate and luciferase expression was immediately confirmed upon addition of D-luciferin substrate (150 μg/mL final concentration). Luciferase bioluminescence was quantified using the Xenogen IVIS-100 and Living Image software (Caliper Life Sciences). GFP mean fluorescence intensity (MFI) vs. luciferase total flux (p/s = photons/second) was plotted and linear regression calculated for the line of best fit (n = 3). All statistical analyses were performed using GraphPad Prism 5.01.