Figure Legends
Figure 1: Comparison of non-viral gene delivery vehicles in Jurkat T cells, with the lead vehicle (Lipo-Hi) represented by black bars. Each vehicle was used to deliver either the luciferase expression plasmid pGL4.50 (A) or the EGFP expression plasmid pEF-GFP (B-F). Panels D-F show representative histograms (cell count vs. cell fluorescence, FL1-H) for Turbofect and 2 concentrations of Lipofectamine. Gates used to calculate %EGFP+ cells are shown as horizontal bars near the top of each panel. Panel C shows the effects of lead vehicles from (A) on cell metabolic activity as measured with MTT as an indication of overall cell viability. Statistically similar cohorts are grouped with letters in (A) and (B), while an asterisk (*) indicates significantly lower values than the control (p<0.05) in (C).
Figure 2: Comparison of common promoters (EF1α, CAG, and CMV) in Jurkat T cells. (A) Expression of luciferase (LUC) from each promoter following delivery with either Lipofectamine (Lipo-Hi) or jetPEI (N:P = 5:1). (B) Transfection efficiencies (%EGFP+
Figure 3:  Lipofectamine volume (µL) impact on transfection efficiency and viability in Jurkat cells. (A) Transfection efficiency at 24 and 48 hours post-transfection and (B) Annexin V/PI viability staining at 24 hours post-transfection. (C) Comparison of the effects of media type (RPMI-1640 and X-VIVOTM
Figure 4: Lipofection of Primary CD3+ T cells. (A) Transfection efficiency of Jurkats and primary CD3+ T cells at optimized conditions (2.75 µL Lipo, 1 µg pEF-EGFP, X-VIVO media, transfected 24 hours post-passage and 30 min after activation). (B) Transfection efficiency of CD3+T cells at different days post-activation with Dynabeads. (C) Representative FACS histogram denoting untransfected and transfected Jurkat T cells.
Figure 5: Flow cytometry analysis of PC-3, Jurkat, and CD3+ primary T cells transfected with either pEF-GFP or a fluorescein-labeled plasmid. (A) Percentage of fluorescent cells 24 hours transfection with either pEF-GFP or the fluorescein-labeled plasmid (B) Mean fluorescence of transfected cells 24 hours post-transfection. (C) Effects of trypsinization on the percentage of fluorescein+ cells and their (D) mean fluorescence levels. Error bars denote one standard deviation. For (A) and (B), CRD ANOVA and Kruskal-Wallis test used to calculate significance within cell lines. For (C) and (D), significance calculated within cell lines using CRD ANOVA and Tukey post-hoc or Kruskal-Wallis test, across time points.
Figure 6: Confocal microscopy images of PC-3 and CD3+ primary T cells transfected with pEF-EGFP and Fluorescein fluorescent plasmid. (A) Confocal microscopy images of PC-3 cells transfected with pEF-EGFP and Fluorescein at 48 hours post-transfection (B) Confocal microscopy images of CD3+ primary T cells at 24 hours post-transfection. (C) Confocal microscopy images/Z-stacks of PC-3 and Primary T cells transfected with fluorescein-labeled pDNA and then stained with Hoescht 33342 and CellBrite Red.