Bioluminescent calcium mediated detection of nanosecond electroporation: Grasping the differences between 100 ns and 100 µs pulses

Abstract

Electroporation is a phenomenon of transient or irreversible permeabilization of the cell membrane after pulsed electric field treatment. Fluorescent probes are frequently used to assess the extent of permeabilization, however, as an alternative, a D-luciferin oxidation-based method can be used. In this work, we have used sequences of a microsecond (1.3 kV/cm × 100 µs) and nanosecond (12.5 kV/cm × 100 ns) pulses to trigger various levels of cell permeabilization and assessed the differences in the response using a conventional fluorescent probe (YO-PRO-1 (YP)) and D-luciferin oxidation methodology. The nanosecond pulses (n = 5-100) have been delivered with 1 kHz repetition frequency, and the results were compared with 1 MHz protocols. Additionally, the effects of extracellular Ca²⁺ have been assessed. Various concentrations of CaCl₂ (2, 5, and 10 mM) have been used, and it was shown that the bioluminescence of the cells after electroporation depends on extracellular calcium concentration. It was shown that the changes in bioluminescence signal could be used as a marker of cell membrane permeabilization on par with YP assay when calcium is added and thus, effectively employed for analysis of electroporation phenomenon in vitro both for nanosecond and microsecond pulses.

Keywords: Bioluminescence; Calcium electroporation; Kinetic measurement; Membrane permeabilization; nsPEF.