<p><i>Panel a</i> 293T cells were transduced with increasing dilutions (different MOIs as indicated) of lentivirus encoding the PS11-scFvFc-CD28-gp41-IRES-ZsGreen. Transduced cells were harvested, stained for Fc-surface expression, and analyzed by FACS. Expression of ZsGreen was measured to monitor levels of transduction. The graphs depict the percentage of transduced cells that express ZsGreen (blue diamonds) and the percentage values of transduced cells that express PS11-scFvFc as monitored by staining with APC-conjugated anti-human Fc IgG (pink squares). <i>Panels b</i> and <i>c</i>, cell -surface expressed scFvFc proteins are functional. 293T cells were transduced with a lentivirus encoding PS11-scFv-Fc-CD28-gp41-IRES-ZsGreen (<i>Panel b</i>) or 11A-scFvFc-CD28-gp41-IRES-ZsGreen (<i>Panel c</i>). Cells were incubated with biotinylated GD03-Fc, a specific antigen for 11A-scFvFc, and stained for streptavidin-APC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003181#s4" target="_blank">Methods</a>, and then analyzed by FACS. Note that two clusters of cells in <i>Panel c</i> represent high (R2 gated) and low (R3 gated) levels of 11A-scFvFc on their surface as measured by APC staining. These could reflect variations in cell-surface expression levels resulting from multiple integration events of the scFvFc cassette following transduction, or the difference in transgene integration site, <i>i.e.</i>, its proximity to active transcriptional units. R2 = 1390 and R3 = 220 are MFI values of 11A scFvFc expressing cells, where percentage of positive cells in each gate is 31% and 49% respectively. <i>Panel d.</i> a summary of specific antigen binding by the scFvFc displayed on the lentivirus transduced cells. The table shows the percentage of transduced cells expressing X48-scFvFc-CD28-gp41 or PS11 scFvFc-CD28-gp41 that bind to their cognate or irrelevant biotinylated antigens as visualized by APC staining and their corresponding MFI values.</p
