An Electroporation Cytometry Protocol for Live-Cell, Fluorescence Microscopy Using U2 OS Cell Culture

Pulsed electric fields (PEFs) have a wide range of applications in medical research and clinical applications. A key area of research focuses on electroporation (reversible or irreversible). Reversible electroporation has been used for several decades for transferring molecules through cell membranes such as plasmid DNA, typically referred to as gene electrotransfer (GET). Conversely, irreversible electroporation has become a popular technique in cancer treatment, providing a non-toxic alternative by permanently rupturing cells using PEFs. This can be combined with various types of drugs or reagents to enhance the effect which has become known as electrochemotherapy (ECT). However, despite the broad success in practical applications, further supporting technology and research is required to improve current techniques. To address this, an electroporation cytometry system was developed to support live-cell PEF experimentation with the ability to perform long-term fluorescence microscopy. This was in combination with a stably expressing FUCCI(CA)5 U2-OS cell line for assessing changes in the cell cycle in response to PEF exposure. In doing so, up to 30 h timelapse microscopy was achieved, providing real-time changes in cell activity based on the FUCCI(CA)5 reporter. As a result, phase-specific changes were quantifiable for downstream analysis.