Probing Nanoelectroporation and Resealing of the Cell Membrane by the Entry of Ca and Ba 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, Cd, Zn, and Ba 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 and Ba 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 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 entry could elicit calcium-induced calcium release (CICR) followed by Ca removal from the cytosol, which markedly affected the time course, polarity, amplitude, and the dose-dependence of fluorescence change. Both Ca and Ba proved as sensitive nanoporation markers, with Ba being more reliable for monitoring membrane damage and resealing.