PMC

Cell survival and permeabilization of C2C12 cells after EP. Survival and permeabilization curves of the cells treated with different number of pulses at different amplitude per distance ratios, 5 ms duration and 1 Hz repetition frequency are presented.

Cytotoxic effects of antioxidants (vitamin C and tempol) on C2C12 cells. Cell survival was determined after exposure of cells to different concentrations of antioxidants (a) and combined with EP and pEGFP-N1 (EGT–EP+pEGPF-N1) (b). Pulsing parameters: 6 × 500 Vcm⁻¹, 5 ms, 1 Hz. Data represent arithmetic mean±s.e.m. of three experiments normalized to untreated control group or to the group exposed only to EP or EGT, where EP or EGT is present.

EP-induced ROS on C2C12 cells which are scavenged by plasmid DNA (pEGFP-N1) or antioxidants (tempol, vitamin C). Lucigenin chemiluminescence measured after electrotransfection or EP (pulsing parameters: 6 pulses, 5 ms, 1 Hz) (a) and after EP with the addition of antioxidants (pulsing parameters: 6 × 500 Vcm⁻¹, 5 ms, 1 Hz) (b). Data represent the arithmetic mean±s.e.m. of 3–5 experiments normalized to untreated control group and cell viability. P<0.05 was considered statistically significant. ^*Statistically different from groups 0–300 Vcm⁻¹ and 500–600 Vcm⁻¹, including the same groups with plasmid DNA, ^**Statistically different from all groups, including the same group with plasmid DNA except 600 Vcm⁻¹, ^***Statistically different from all groups, including the same group with plasmid DNA except 500 and 700 Vcm⁻¹, ^****Statistically different from groups 0–300 Vcm⁻¹, including the same group with plasmid DNA, ^#Statistically different from groups 0 Vcm⁻¹ with plasmid DNA, ^##Statistically different from groups 0–100 Vcm⁻¹ with plasmid DNA, ^###Statistically different from groups 0–600 Vcm⁻¹ with plasmid DNA.

The integrity of plasmid DNA (pEGFP-N1) and genomic DNA damage after EP. Representative image of agarose gel electrophoresis of plasmid DNA (pEGFP-N1) after EP or the addition of bleomycin (a) and normalized fluorescence intensity of FITC conjugate bound to 8-oxoguanine after EP indicative of oxidative DNA damage (b). Pulsing parameters: 6 × 400–600 Vcm⁻¹, 5 ms, 1 Hz for (a) and 6 × 500 Vcm⁻¹, 5 ms, 1 Hz for (b). Groups: 0 V—plasmid DNA (pEGFP-N1), 400 V—plasmid DNA (pEGFP-N1)+EP 400 Vcm⁻¹, 500 V—plasmid DNA (pEGFP-N1)+EP 500 Vcm⁻¹, 600 V—plasmid DNA (pEGFP-N1)+EP 600 Vcm⁻¹, bleomycin 1, 10, 100 μ—plasmid DNA (pEGFP-N1)+bleomycin. Data in (b) represent the arithmetic mean±s.e.m. of three experiments normalized to the untreated control group: EP—electropermeabilization, DNA—pEGFP-N1 plasmid.

Increased transfected area (area) and mean fluorescence of transfected area (mean fluorescence) of electrotransfected M. tibialis cranialis after the addition of tempol. Transfected area and mean fluorescence intensity were determined by analysis of the acquired images of M. tibialis cranialis (a) and imaged with a fluorescent stereomicroscope (excitation: 470/40 nm, emission: 525/50 nm) (b). Pulsing parameters: 1 × 600 Vcm⁻¹, 100 μs, 1 Hz+4 × 80 Vcm⁻¹, 100 ms, 1 Hz. Groups in (b): plasmid+EP–control, plasmid+EP+tempol 6 m—tempol 6 m, plasmid+EP+tempol 12 m–tempol 12 m, plasmid+EP+tempol 24 m—tempol 24 m. Data in (a) represent the arithmetic mean±s.e.m. of 13–35 mice legs normalized to the control group in which no antioxidants were added. P<0.05 was considered statistically significant. ^*Statistically different from all other groups.

Genomic DNA damage after EP. Genomic DNA damage was determined by modified Comet assay with repair enzyme Fpg after the exposure of cells to EP or tert-butyl hydroperoxide. The results are presented as a percentage of tail DNA. Data are presented as quantile box plots. The edges of the box represent the 25 and 75th percentiles (95% confidence intervals), the mean is a solid line through the box. The white box plots represent exposure to enzyme buffer (DNA-strand breaks), while dotted box plots represent exposure to Fpg enzyme (oxidized purines). Pulsing parameters: 6 × 500 Vcm⁻¹, 5 ms, 1 Hz. Groups: 0 buffer—negative control group without enzyme treatment; ^*statistically different from the control group with the same treatment, ⁺statistically significantly different between buffer exposed groups and Fpg enzyme exposed groups.

Increased in vitro transfection efficiency of pEGFP-N1 plasmid into C2C12 cells after the addition of tempol before EP. Fraction of enhanced GFP expressing cells (a) and normalized median fluorescence intensity of enhanced GFP expressing cells (b). Images of cells electrotransfected with pEGFP-N1 plasmid acquired with fluorescent microscope (c) (excitation: 460–490 nm, emission: 505 nm). Pulsing parameters: 6 × 500 Vcm⁻¹, 5 ms, 1 Hz. Data in (a) and (b) represent arithmetic mean±s.e.m. of three to five experiments normalized to control group in which no antioxidants were added. P<0.05 was considered statistically significant. ^*Statistically different from control group and the same concentration of tempol after EP, ^**Statistically different from control group, all tempol before EP groups except tempol 6 m and from tempol 8 m after EP. Groups in (c): plasmid+EP—EGT, pEGFP-N1 plasmid+EP+tempol 8 m—EGT+tempol 8 m, plasmid+EP+tempol 10 m—EGT+tempol 10 m, addition of antioxidant after EP—antioxidant after EP, addition of antioxidant before EP—antioxidant before EP. Where no antioxidant was added, the treatment of cells was the same for both parallel groups.