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Published on: February 8, 2015
An Electroporation Cytometry Protocol for Live-Cell, Fluorescence Microscopy Using U2 OS Cell Culture
Thomas Nesmith1, Christian Vieira2, Darius G Rackus1
1Department of Chemistry and Biology, Toronto Metropolitan University; Institute for Biomedical Engineering, Science and Technology (iBEST), a partnership between St. Michael's Hospital, a site of Unity Health Toronto, and Toronto Metropolitan University; Keenan Research Centre for Biomedical Science at St. Michael's Hospital.
Pulsed electric fields (PEFs) enable gene electrotransfer and cancer treatment. A new system allows live-cell microscopy to track cell cycle changes after PEF exposure, improving electroporation techniques.
Area of Science:
- Biomedical Engineering
- Cell Biology
- Medical Physics
Background:
- Pulsed electric fields (PEFs) are crucial in medical research, particularly for electroporation techniques like gene electrotransfer (GET) and electrochemotherapy (ECT).
- While effective, current electroporation methods require advanced supporting technologies for detailed live-cell analysis and improved efficacy.
Purpose of the Study:
- To develop an advanced electroporation cytometry system for live-cell experimentation with long-term fluorescence microscopy.
- To assess real-time cell cycle changes in response to PEF exposure using a FUCCI(CA)5 U2-OS cell line.
Main Methods:
- Development of an electroporation cytometry system integrated with long-term fluorescence microscopy capabilities.
- Utilized a stably expressing FUCCI(CA)5 U2-OS cell line to monitor cell cycle dynamics.
- Conducted timelapse microscopy for up to 30 hours to observe cellular responses to PEF exposure.
Main Results:
- Achieved up to 30 hours of timelapse microscopy, enabling real-time observation of cell activity.
- Quantified phase-specific changes in the cell cycle in response to PEF treatment.
- Demonstrated the system's capability to support detailed live-cell PEF experimentation.
Conclusions:
- The developed electroporation cytometry system effectively supports live-cell PEF experimentation and long-term monitoring.
- This technology provides a valuable tool for quantifying PEF-induced cell cycle alterations.
- The findings contribute to the advancement of electroporation techniques in biomedical research.
