Electroporation of Ishikawa cells: analysis by flow cytometry

Abstract

Electroporation facilitates loading of cells with molecules and substances that are normally membrane impermeable. Flow cytometry is used in this study to examine the effects of the application of electroporation-level monopolar electric field pulses of varying electrical field strength on Ishikawa endometrial adenocarcinoma cells. Analysis of the fluorescence versus forward scatter plots corroborates the well-recognised threshold and cell size dependence characteristics of electroporation, but also shows the progression of cell lysis and generation of particulate material. Two 500 µs monopolar rectangular pulses ranging from 1.0 × 10⁵ to 2.5 × 10⁵ V/m were used to electroporate the cells. Electroporation yields (fraction of viable cells exhibiting significant propidium iodide uptake) ranged from 0 to 97%, with viability ranging between 78 and 34% over the electric field strength range tested. The higher electric field strength pulses not only reduced cell viability, but also generated a substantial amount of sub-cellular sized particulate material indicating cells have been physically disrupted enough to create these particles.

Fig. 1

Two typical flow cytometer raw data logarithmic‐linear plots, showing fluorescence versus forward scatter (a) Dot plot of a negative control set where no electroporation treatment was applied, (b) Dot plot of a sample set receiving electroporation treatment of two monopolar 1.5 × 10⁵ V/m pulses of 500 µs duration, separated by an interval of 1 s. Both samples were chemically and physically treated in an identical manner. Features are identified in enclosed regions that represent sub‐populations of interest. Note: The absolute flow cytometry count is given at the end of each sub‐population label. These counts are relative to the total count of 30,000 particles (3 sets of 10,000 particles were combined) for each graph

Fig. 2

Superimposed dot plots of a control set (blue dots) and an electroporation set (1.5 × 10⁵ V/m pulses – overlaid red dots), enabling visual comparison between the effect of significant electroporation to a negative control. These plots correspond with the conditions of Fig. 1

Fig. 3

Electroporation effect trends (as relative percentages) associated with the data used to derive Fig. 6. Error bars are shown for p < 0.05

Fig. 4

Raw dot plots at pulses of increasing field intensities (a) Dot plot for 1.5 × 10⁵ V/m pulses with the area enclosed by the dashed brown line showing a low number of cells in the transitional non‐viable cells sub‐population (1.7% of the total population of cells counted), (b) Dot plot for 2.5 × 10⁵ V/m pulses with the area enclosed by the solid brown line showing a high number of cells in the transitional non‐viable cells sub‐population (13.7% of the total population of cells counted)

Fig. 5

Typical maximum cell density plot for three electroporated sample sets, for 1.5 × 10⁵ V/m pulses. The green data points represent maximum cell density for the forward scatter values shown. A least mean squares linear trend line has been added (green line), with the accompanying R‐squared value as a confidence metric. Error bars are shown for p < 0.05

Fig. 6

Maximum cell density plots for sets exposed to electric field pulses ranging from 1.0 × 10⁵ to 2.5 × 10⁵ V/m, including a negative control. Error bars are shown for p < 0.05