Figure 3
Figure 3. Cell separation efficiency and specific transfection/transduction of the target cells using nonviral and viral magselectofection Jurkat T cells as a target. (A) A mixture of 2.5 × 106 Jurkat T cells and 2.5 × 106 K562 cells was treated with CD2 MicroBeads and passed (i) sequentially through 2 LS columns (MACS procedure) or (ii) through one LS column, followed by magselectofection at the second LS column modified with PEI-Mag2/DF-Gold/pBLuc magnetic lipoplexes composed of 20 μg plasmid DNA (DNA/DF-Gold/MNP = 1:4:1; weight/volume/Fe weight). The CD2-cell fraction in the effluent (K562 cells) and the CD2+ cells positively selected in the column (Jurkat T cells) were treated with a CD3-PE antibody and analyzed for the percentage of CD3-PE+ cells using FACS analysis. Dot plots show the data for single measurement and the percentage of CD3+ cells (mean ± SD from triplicates in the inset boxes). (B) Luciferase expression in the effluent (CD3−/CD2− cells) and in the cell fraction that was magnetically selected with CD2 beads (CD3+/CD2+ cells). (C) A mixture of 0.5 × 106 Jurkat T cells and 0.5 × 106 K562 cells was treated with CD2 MicroBeads and passed (i) sequentially through 2 LS columns (MACS procedure) and (ii) through one LS column, followed by magselectofection in the second LS column modified with SO-Mag2/LV.eGFP magnetic lentivirus complexes (0.5 × 106 TU/column, 20 fg Fe/VP). The CD2− cell fraction in the effluent (K562 cells) and the CD2+ cells positively selected within the column (Jurkat T cells) were treated with a CD3-PE antibody and analyzed for the percentage of CD3-PE+ cells using FACS analysis. Dot plots show the data 31 for single measurement, and the percentage of CD3+ cells is given as mean plus or minus SD from triplicates in the inset boxes. (D) The cell fractions in the effluent and the CD2− selected fraction after the second column loaded with SO-Mag2/LV.eGFP magnetic lentivirus complexes formulated at 20 fg Fe/VP were analyzed for eGFP expression using FACS analysis. FACS data are shown for MOI of 2. The right graph shows the percentages of eGFP+ cells in the effluent and in the CD2-selected fraction at different time points after magselectofection using an MOI of 0.5. (E) K562 cells as a target. A mixture of 9 × 106 Jurkat T cells and 1 × 106 K562 was labeled with CD2 MicroBeads for depleting the Jurkat T cells on an unmodified LS column. The cells in the effluent were labeled with CD33 MicroBeads and applied to the second vector-loaded LS column (SO-Mag2/LV.eGFP magnetic lentivirus complexes, 2 × 106 TU/column, formulated at 10 fg Fe/VP). The CD2-selected cells from the first column were analyzed for CD2 expression and the CD33-selected and effluent fractions from the second column were analyzed for eGFP expression using FACS analysis. All experiments were carried out in triplicate.

Cell separation efficiency and specific transfection/transduction of the target cells using nonviral and viral magselectofection Jurkat T cells as a target. (A) A mixture of 2.5 × 106 Jurkat T cells and 2.5 × 106 K562 cells was treated with CD2 MicroBeads and passed (i) sequentially through 2 LS columns (MACS procedure) or (ii) through one LS column, followed by magselectofection at the second LS column modified with PEI-Mag2/DF-Gold/pBLuc magnetic lipoplexes composed of 20 μg plasmid DNA (DNA/DF-Gold/MNP = 1:4:1; weight/volume/Fe weight). The CD2-cell fraction in the effluent (K562 cells) and the CD2+ cells positively selected in the column (Jurkat T cells) were treated with a CD3-PE antibody and analyzed for the percentage of CD3-PE+ cells using FACS analysis. Dot plots show the data for single measurement and the percentage of CD3+ cells (mean ± SD from triplicates in the inset boxes). (B) Luciferase expression in the effluent (CD3−/CD2− cells) and in the cell fraction that was magnetically selected with CD2 beads (CD3+/CD2+ cells). (C) A mixture of 0.5 × 106 Jurkat T cells and 0.5 × 106 K562 cells was treated with CD2 MicroBeads and passed (i) sequentially through 2 LS columns (MACS procedure) and (ii) through one LS column, followed by magselectofection in the second LS column modified with SO-Mag2/LV.eGFP magnetic lentivirus complexes (0.5 × 106 TU/column, 20 fg Fe/VP). The CD2− cell fraction in the effluent (K562 cells) and the CD2+ cells positively selected within the column (Jurkat T cells) were treated with a CD3-PE antibody and analyzed for the percentage of CD3-PE+ cells using FACS analysis. Dot plots show the data 31 for single measurement, and the percentage of CD3+ cells is given as mean plus or minus SD from triplicates in the inset boxes. (D) The cell fractions in the effluent and the CD2− selected fraction after the second column loaded with SO-Mag2/LV.eGFP magnetic lentivirus complexes formulated at 20 fg Fe/VP were analyzed for eGFP expression using FACS analysis. FACS data are shown for MOI of 2. The right graph shows the percentages of eGFP+ cells in the effluent and in the CD2-selected fraction at different time points after magselectofection using an MOI of 0.5. (E) K562 cells as a target. A mixture of 9 × 106 Jurkat T cells and 1 × 106 K562 was labeled with CD2 MicroBeads for depleting the Jurkat T cells on an unmodified LS column. The cells in the effluent were labeled with CD33 MicroBeads and applied to the second vector-loaded LS column (SO-Mag2/LV.eGFP magnetic lentivirus complexes, 2 × 106 TU/column, formulated at 10 fg Fe/VP). The CD2-selected cells from the first column were analyzed for CD2 expression and the CD33-selected and effluent fractions from the second column were analyzed for eGFP expression using FACS analysis. All experiments were carried out in triplicate.

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