(a) Scheme of the vectors that mediate co-expression of iRFP720 and firefly luciferase either via IRES- or E2A-mediated mechanisms (not drawn to scale). Arrows above and below the scheme indicate the reporter gene transcript and open reading frames, respectively. (b) FACS analyses of iRFP720 fluorescent signal intensity in purified mMSC populations, which had been transfected with either the IRES or E2A vector, respectively, indicated similar levels of expression of the first ORF. (c) Confocal fluorescence microscopy of transfected cells for co-expression of iRFP720 and luciferase (immunofluorescence). In contrast to Luc, iRFP720 tends to accumulate in the nucleus (yellow triangles). (d, e) Comparison of fluorescent and bioluminescent signal intensities from IRES and E2A vector-transfected cell populations using IVIS Spectrum imaging. Although both cell suspensions provided very similar levels of fluorescence from the first reporter ORF (iRFP720) in the region of interest (ROI), bioluminescence levels were 5-fold higher in the E2A vector-transfected cell population, indicating a more efficient translation of the second ORF (luciferase) from the E2A element than from the IRES. Fluorescence was measured first, followed by addition of CycLuc1 to the same cell suspensions and bioluminescence imaging.