Figure 2.

Validation of qPCR method for intracellular pDNA copy number measurement. (A) Typical image of agarose gel showing pDNA and its degraded fragments. Gel electrophoresis was performed after pDNA solutions prepared with complete culture medium were treated with the DRP diluted by different factors (1:40, 1:160, 1:320, and 1:640). The pDNA solutions in untreated control groups were prepared with pure water or complete culture medium (Med). (B)-(E) Typical fluorescence images of pDNA (red) and cells (green) after the buffer containing Rho-pDNA and cells (stained with CellTracker™ Green CMFDA) was treated with one electric pulse (650 V and 400 μs) (Pulsed). To remove extracellular pDNA after electrotransfection, the cells were treated with the DRP (1:100 dilution). The non-treated groups served as controls. (F)-(I) Typical fluorescence images of Rho-pDNA and cells. The experimental condition was similar to that in Panels B-E, except that the pulsing buffer was supplemented with 0.2% type B gelatin, and that 3 pulses (650 V/cm, 400 μs, 2 s interval) were applied to the cells. (J)&(K) Images of Rho-pDNA and cells at a higher magnification. The samples were from the same groups as those shown in Panels G&I, respectively. These images show apoptotic cells (black arrows) and pDNA aggregates either alone or complexed with membrane debris (white arrowheads). Scale bars: 40 μm in B-I; 20 μm in J&K.