Calibration of the propidium iodide signal

To identify the propidium iodide fluorescence value that corresponded to 100% cell death we exposed KLN 205 embedded in 1% agarose to increasing numbers of 300 ns pulses at 600 V (Fig. 2). At 2 h after the exposure, we stained both viable and dead cells. Significant cell killing was detected after 300 or more pulses, both as a decrease in tetrazolium salt reduction and as an increase in PI uptake (Fig. 2A). To quantify this effect, we measured the PI fluorescence intensity in the region between the electrodes as shown in Fig. 2A. The PI fluorescence intensity reached a plateau at 500–1000 pulses indicating that at these doses 100% of the cells in the quantified area were killed (Fig. 2B). Therefore, in subsequent experiments a parallel control exposure to 3000 pulses was used as a reference point for PI expression that corresponds to 100% cell death. A similar calibration was done for the spheroids exposed in Matrigel and the plateau was reached at 1000–2000 pulses (data not shown).