Figure 2. Cooling after nsEP exposure delays membrane resealing causing cell swelling.

U-937 cells were exposed to 50, 300 ns pulses (100 Hz, 7 kV/cm) at room temperature, followed by incubation either on ice or at 37 °C. The medium containing PI was added either immediately after nsEP (“time 0”), or after 10 or 30 min of incubation at different temperatures. (A) Effect of post-nsEP incubation time and temperature on PI uptake and cell diameter in individual cells. Data for samples not treated with nsEP (sham-exposed negative control) are shown in black in all panels. For a positive control, cells were permeabilized with 40 μg/ml digitonin for 5 min. Horizontal dashed lines show the fluorescence threshold to identify PI-positive cells. (B) The fraction of PI-positive cells after different treatments. Mean +/− s.e. for n = 3, *p < 0.001 for the effect of cooling vs 37 °C. (C) Post-nsEP cooling for 30 min causes cell swelling (right image and histogram). Filled bars in the histogram show the distribution of cell diameters in sham-exposed control samples. Cells in left panels (nsEP followed 30 min at 37 °C) were not different in appearance or size from the controls. Scale bar: 50 μm. The histogram data are 300–500 cells measured per sample from 3 independent experiments.