Probing Nanoelectroporation and Resealing of the Cell Membrane by the Entry of Ca(2+) and Ba(2+) Ions

The principal bioeffect of the nanosecond pulsed electric field (nsPEF) is a lasting cell membrane permeabilization, which is often attributed to the formation of nanometer-sized pores. Such pores may be too small for detection by the uptake of fluorescent dyes. We tested if Ca(2+), Cd(2+), Zn(2+), and Ba(2+) ions can be used as nanoporation markers. Time-lapse imaging was performed in CHO, BPAE, and HEK cells loaded with Fluo-4, Calbryte, or Fluo-8 dyes. Ca(2+) and Ba(2+) did not change fluorescence in intact cells, whereas their entry after nsPEF increased fluorescence within <1 ms. The threshold for one 300-ns pulse was at 1.5–2 kV/cm, much lower than >7 kV/cm for the formation of larger pores that admitted YO-PRO-1, TO-PRO-3, or propidium dye into the cells. Ba(2+) entry caused a gradual emission rise, which reached a stable level in 2 min or, with more intense nsPEF, kept rising steadily for at least 30 min. Ca(2+) entry could elicit calcium-induced calcium release (CICR) followed by Ca(2+) removal from the cytosol, which markedly affected the time course, polarity, amplitude, and the dose-dependence of fluorescence change. Both Ca(2+) and Ba(2+) proved as sensitive nanoporation markers, with Ba(2+) being more reliable for monitoring membrane damage and resealing.