Journal: Cytotechnology

Article Title: Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified, long shelf-life polyethylenimine

doi: 10.1007/s10616-010-9259-z

Figure Lengend Snippet: Increased transfection efficiency by acidic DNA compaction. PEI and pcDNA4-TO-EGFP were mixed in HBS (pH 7.4) or LBS (pH 3.5) [PEI/DNA ratio = 5 (μg/μg)], and the resulting PEI/DNA polyplex was transiently transfected into HeLa cells. At 24 h posttransfection, EGFP expression was analyzed by flow cytometry. a 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). Transfection efficiencies were quantitated by counting the number of cells expressing EGFP at high levels and at low to high levels. b HeLa cells transiently transfected with the polyplex formed in HBS (pH 7.4) or LBS (pH 3.5) were stained with PI and analyzed for PI-stained dead cells by flow cytometry. Data represent means ± SD (n = 3)

Article Snippet: Cell lines Human epithelial HeLa and HeLa S3 cells (Japanese Collection of Research Bioresources, Osaka) were cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 5% fetal bovine serum (FBS) and with 5% bovine serum (BS), respectively.

Techniques: Transfection, Expressing, Flow Cytometry, Control, Staining

Journal: Cytotechnology

Article Title: Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified, long shelf-life polyethylenimine

doi: 10.1007/s10616-010-9259-z

Figure Lengend Snippet: 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)

Article Snippet: Cell lines Human epithelial HeLa and HeLa S3 cells (Japanese Collection of Research Bioresources, Osaka) were cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 5% fetal bovine serum (FBS) and with 5% bovine serum (BS), respectively.

Techniques: Transfection, Staining, Flow Cytometry, Expressing, Control

Journal: Cytotechnology

Article Title: Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified, long shelf-life polyethylenimine

doi: 10.1007/s10616-010-9259-z

Figure Lengend Snippet: Transfection into COS-1, HeLa S3, Dami, and HCT116 cells. a–c EGFP expression was examined by fluorescence microscopy. Scale bars, 10 μm. a COS-1 and HeLa S3 cells were transiently transfected with the polyplex formed by mixing 5 μg of pcDNA4-TO-EGFP with 25 μg of PEI in LBS (pH 4.0), and after 12 h of culture the medium was replaced with fresh serum-containing medium. b COS-1 cells were transiently transfected with the polyplex formed by mixing 1 μg of pcDNA4-TO-EGFP with 5 μg of PEI in LBS (pH 4.0) or an optimal amount of TransIT-LT1. c Dami cells in suspension culture were attached to culture dishes, as described under “Materials and methods”, and transiently transfected with the polyplex formed by mixing 4 μg of pcDNA4-TO-EGFP with 26 μg of PEI in LBS (pH 4.0). d HCT116 cells were transiently transfected once or twice with the polyplex formed by mixing 6 μg of pCAG/TetR-IRES-CD25 with 7.5 μg of PEI in LBS (pH 4.0), as described under “Materials and methods”, and stained with anti-CD25 antibody. Representative histograms of control (shaded areas) and transfected cells (solid line) are shown for CD25 expression at low to high levels (thin arrows). e HeLa S3 cells were stably transfected with the polyplex formed by mixing pcDNA4-TO-EGFP with PEI in LBS, as described under “Materials and methods”. Zeocin-resistant colonies were cloned in 2 weeks and observed by fluorescence microscopy. Scale bar, 10 μm

Article Snippet: Cell lines Human epithelial HeLa and HeLa S3 cells (Japanese Collection of Research Bioresources, Osaka) were cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 5% fetal bovine serum (FBS) and with 5% bovine serum (BS), respectively.

Techniques: Transfection, Expressing, Fluorescence, Microscopy, Suspension, Staining, Control, Stable Transfection, Clone Assay