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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 &lt;1 ms. The threshold for one 300-ns pulse was at 1.5-2 kV/cm, much lower than &gt;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.

Probing Nanoelectroporation and Resealing of the Cell Membrane by the Entry of Ca2+ and Ba2+ Ions