Fig. 2.

Effects of PEI/DNA ratios and pH of compaction medium on transfection efficiency. a, b PEI and pcDNA4-TO-EGFP were mixed in LBS (pH 3.5) at various PEI/DNA ratios (μg/μg), and the resulting PEI/DNA polyplex was transiently transfected into HeLa cells. a At 24 h posttransfection, cells were stained with PI for dead cells, and analyzed by flow cytometry for EGFP expression (filled bars) and cytotoxicity (open bars). Data represent means ± SD (n = 3). b PEI and pcDNA4-TO-EGFP were mixed in LBS (pH 3.5) at a ratio of 6 (μg/μg). Representative histograms of control (shaded areas) and transfected cells (solid line) are shown. Two gates indicate EGFP expression at high levels (thick arrows) and at low to high levels (thin arrows). c PEI and pcDNA4-TO-EGFP were mixed at a ratio of 5 (μg/μg) in HBS (pH 7.4) or LBS (pH 3.5, 4.0, 4.5), and the resulting PEI/DNA polyplex was transiently transfected into HeLa cells. Transfection efficiencies were quantitated by counting the number of cells expressing EGFP at high levels (filled bars) and at low to high levels (open bars). Results are expressed as values relative to the number of cells expressing EGFP using HBS as DNA compaction medium (pH 7.4). Data represent means ± SD (n = 3). Transfection efficiencies were 2.5 and 16.5% at pH 7.4 and pH 4.0, respectively in cells expressing EGFP at high levels. d, e Five microgram of new or old PEI and 1 μg of pcDNA4-TO-EGFP were mixed in LBS (pH 4.0), and 5 μg of new PEI and 5 μg of old PEI (mix) and 1 μg of pcDNA4-TO-EGFP were mixed in LBS (pH 4.0). The resulting PEI/DNA polyplex was transiently transfected into HeLa S3 cells, and transfection efficiencies were quantitated by counting the number of cells expressing EGFP. Data represent means ± SD (n = 3)