International Journal of Molecular Sciences
3386 (17 pp.)
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 Ca2+, Cd2+, Zn2+, and Ba2+ 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. Ca2+ and Ba2+ 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. Ba2+ 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. Ca2+ entry could elicit calcium-induced calcium release (CICR) followed by Ca2+ removal from the cytosol, which markedly affected the time course, polarity, amplitude, and the dose-dependence of fluorescence change. Both Ca2+ and Ba2+ proved as sensitive nanoporation markers, with Ba2+ being more reliable for monitoring membrane damage and resealing.
Original Publication Citation
Bo, W., Silkunas, M., Mangalanathan, U., Novickij, V., Casciola, M., Semenov, I., ... & Pakhomov, A. G. (2020). Probing nanoelectroporation and resealing of the cell membrane by the entry of Ca2+ and Ba2+ Ions. International Journal of Molecular Sciences, 21(9), 3386. doi: 10.3390/ijms21093386
Bo, Wenfei; Silkunas, Mantas; Mangalanathan, Uma; Novickij, Vitalij; Casciola, Maura; Semenov, Iurii; Xiao, Shu; Pakhomova, Olga N.; and Pakhomov, Andrei G., "Probing Nanoelectroporation and Resealing of the Cell Membrane by the Entry of Ca2+ and Ba2+ Ions" (2020). Bioelectrics Publications. 285.
0000-0003-4017-8260 (Bo), 0000-0003-0203-0847 (Casciola), 0000-0002-0302-1355 (Semenov), 0000-0003-4950-4130 Pakhomova), 0000-0003-3816-3860 (Pakhomov)