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.