Integrating High-Content Imaging to Investigate Cellular Responses to Toxic Drug Compounds and Electroporation in Monolayer Cultures
College
The Graduate School
Department
Bioelectrics
Graduate Level
Doctoral
Graduate Program/Concentration
Biomedical Sciences
Presentation Type
Poster Presentation
Abstract
Electroporation of cell monolayers preserves cell physiology and eliminates the need for detachment, making it an advantageous approach for studying membrane integrity, cell death, and other processes in electroporated cells. We adapted a previously developed monolayer electroporation methodology (Gudvangen et al., 2022) to assess cellular responses to the combined treatment of a membrane-impermeable ribosome-inactivating toxin and pulsed electric fields (PEF). The method involves applying electric pulses between two cylindrical electrodes, spaced 2.2 mm apart, and inserted vertically into wells of cell culture plate, makingplate, making a gentle contact with the cell monolayer. A customized Anet A8 3D printer (Shenzhen Anet Technology Co., China) was utilized to precisely position electrodes in each well, ensuring consistent PEF exposure and imaging intervals.
The electrodes generated a non-uniform electric field that gradually decayed with distance, allowing efficient testing of a broad range of field strengths within a single PEF-exposed sample. The approach allows for high-content imaging to identify electroporated and dead cells using fluorescent markers such as Hoechst, YoPro-1 (YP), and Propidium iodide (PI) dyes. Hoechst labeled all nuclei, YP marked electroporated cells, and PI identified cells that failed to restore membrane integrity. Imaging was performed with an Olympus IX83 fluorescence microscope (Olympus America, Hamden, CT), equipped with an automated scanning stage, allowing for the imaging of multiple the wells in the cell culture plate in a timely manner. Several adjacent images were captured for the each well and stitched together providing a comprehensive view of all the cells between electrodes. The images were analyzed with ImageJ Fiji image processing program (Rasband et al. 1997-2018). YP up take indicated reversible electroporation, while delayed PI uptake signified cell death. The “all-or-none” PI uptake pattern allowed for precise identification of irreversible electroporation margins and their enhancement by the cytotoxic compound. Changes in these margins in combinatory treatments served as a measure of electroporation-mediated drug toxicity potentiation. This methodology enables the quantification of electroporation thresholds, irreversible electroporation zones, and drug toxicity enhancement. Additionally, it provides insights into the time course and modalities of cell death following PEF exposure both alone and in combination with cytotoxic drugs, offering a powerful approach for studying electroporation and drug cytotoxicity potentiation.
Keywords
Cell monolayer electroporation, Pulse electric field, High-content imaging, Electropermeabilization
Integrating High-Content Imaging to Investigate Cellular Responses to Toxic Drug Compounds and Electroporation in Monolayer Cultures
Electroporation of cell monolayers preserves cell physiology and eliminates the need for detachment, making it an advantageous approach for studying membrane integrity, cell death, and other processes in electroporated cells. We adapted a previously developed monolayer electroporation methodology (Gudvangen et al., 2022) to assess cellular responses to the combined treatment of a membrane-impermeable ribosome-inactivating toxin and pulsed electric fields (PEF). The method involves applying electric pulses between two cylindrical electrodes, spaced 2.2 mm apart, and inserted vertically into wells of cell culture plate, makingplate, making a gentle contact with the cell monolayer. A customized Anet A8 3D printer (Shenzhen Anet Technology Co., China) was utilized to precisely position electrodes in each well, ensuring consistent PEF exposure and imaging intervals.
The electrodes generated a non-uniform electric field that gradually decayed with distance, allowing efficient testing of a broad range of field strengths within a single PEF-exposed sample. The approach allows for high-content imaging to identify electroporated and dead cells using fluorescent markers such as Hoechst, YoPro-1 (YP), and Propidium iodide (PI) dyes. Hoechst labeled all nuclei, YP marked electroporated cells, and PI identified cells that failed to restore membrane integrity. Imaging was performed with an Olympus IX83 fluorescence microscope (Olympus America, Hamden, CT), equipped with an automated scanning stage, allowing for the imaging of multiple the wells in the cell culture plate in a timely manner. Several adjacent images were captured for the each well and stitched together providing a comprehensive view of all the cells between electrodes. The images were analyzed with ImageJ Fiji image processing program (Rasband et al. 1997-2018). YP up take indicated reversible electroporation, while delayed PI uptake signified cell death. The “all-or-none” PI uptake pattern allowed for precise identification of irreversible electroporation margins and their enhancement by the cytotoxic compound. Changes in these margins in combinatory treatments served as a measure of electroporation-mediated drug toxicity potentiation. This methodology enables the quantification of electroporation thresholds, irreversible electroporation zones, and drug toxicity enhancement. Additionally, it provides insights into the time course and modalities of cell death following PEF exposure both alone and in combination with cytotoxic drugs, offering a powerful approach for studying electroporation and drug cytotoxicity potentiation.