Novickij Vitalij, Zinkevičienė Auksė, Radzevičiūtė Eivina, Kulbacka Julita, Rembiałkowska Nina, Novickij Jurij, Girkontaitė Irutė
Faculty of Electronics, Vilnius Gediminas Technical University, Vilnius, Lithuania.
State Research Institute Centre for Innovative Medicine, Department of Immunology, Vilnius, Lithuania.
Bioelectrochemistry. 2022 Jun;145:108084. doi: 10.1016/j.bioelechem.2022.108084. Epub 2022 Feb 4.
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 Ca have been assessed. Various concentrations of CaCl (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.
电穿孔是指细胞在脉冲电场处理后细胞膜出现瞬时或不可逆通透化的现象。荧光探针常被用于评估通透化程度，不过，作为一种替代方法，也可使用基于D - 荧光素氧化的方法。在本研究中，我们使用了微秒级（1.3 kV/cm × 100 µs）和纳秒级（12.5 kV/cm × 100 ns）脉冲序列来引发不同程度的细胞通透化，并使用传统荧光探针（YO - PRO - 1（YP））和D - 荧光素氧化方法评估了响应差异。纳秒级脉冲（n = 5 - 100）以1 kHz的重复频率施加，并将结果与1 MHz的方案进行了比较。此外，还评估了细胞外钙的影响。使用了不同浓度的CaCl₂（2、5和10 mM），结果表明电穿孔后细胞的生物发光取决于细胞外钙浓度。结果显示，当添加钙时，生物发光信号的变化可作为与YP检测相当的细胞膜通透化标志物，因此可有效用于体外分析纳秒级和微秒级脉冲的电穿孔现象。
