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

The scope of this research focuses on the effect of pulsed electric fields on cell behavior, particularly how electroporation affects cell cycle.

Primary advantage of this protocol is the ability to perform continuous live cell electroporation experiments using mammalian cell culture for up to 72 hours. Our lab is focused on adapting these techniques for microfluidics, which will allow for lower sample sizes, reduced reagent requirements, and better sample control for future protocols.

[Narrator] To begin, culture U2OS FUCCI CA5 cells in a 10-centimeter plate with 10 milliliters of DMEM high glucose containing 10% FBS and 1% penicillin streptomycin. Incubate the plate at 37 degrees Celsius in a 5% carbon dioxide environment. When the culture reaches 80% confluency, aspirate the media from the plate. Wash the cells with PBS then add one milliliter of 0.25% trypsin and incubate for 1.5 minutes to dissociate the cells. Neutralize the trypsin by resuspending the dissociated cells in five milliliters of DMEM high glucose. Obtain a 100-microliter sample from the resuspended culture and place it on the bottom glass cover slip inside the electroporation cytometry system chamber. Ensure that the chamber is 30% confluent after introducing the sample. Slowly add four milliliters of DMEM high glucose to the far end of the chamber, preventing the liquid from flushing out the sample. Place a standard microscope slide over the chamber and incubate the chamber at 37 degrees Celsius and 5% carbon dioxide for 24 hours. Turn on the inverted fluorescent microscope with an environmental control chamber to allow the system to stabilize prior to electroporation. While the environmental chamber is stabilizing, use alligator clip cables to connect a digital multimeter to the pulse generator capacitor and high-voltage switch for rapid setup with the electroporation chamber. Now, select 20x magnification on the microscope. In the microscope software, ensure FITC/GFP and A594 Texas Red M Cherry channels are selected corresponding to the two fluorescent proteins in the FUCCI CA5 system. Use low exposure settings, typically less than 0.05 seconds, to prevent photo damage during long-term imaging. Take test images to optimize image focus and quality before initiating the time lapse imaging. Program the time lapse to capture images from three to six different viewpoints every 10 minutes over a 24-hour period to obtain a high volume of cell cycle data per time lapse while mitigating photo damage. Ensure that autofocus is enabled for the A594 channel to optimize the tracking of culture during the time lapse. Leave the software open until electroporation is completed. Place the electroporation cytometry chamber into the microscope equipped with an appropriate environmental chamber. Prior to electroporation, keep the chamber closed and ensure the environmental system remains running to maintain proper cell cycle conditions. Open the environmental chamber and attach a digital multimeter to the pulse generator, then using alligator clip cables, connect the pulse generator to the chamber electrodes. Charge the pulse generator capacitor to 180 volts. Discharge the pulse into the chamber rapidly until the digital multimeter displays zero volts. Remove all electrical connections from the chamber and promptly replace the chamber lid to ensure optimal culturing conditions. Activate the time lapse imaging immediately once the environmental chamber is securely in place. After the time lapse is complete, use the microscope software to save all captured images as a single video. Adjust visual quality to enhance the clarity of cell features for manual quantification. Finally, observe the changes in FITC and A594 signals over time to track cell phases in the time lapse video. Using the Fuji system, Visual data were collected by tracking color changes linked to protein expression and ubiquitination, allowing clear distinction of G1, S, G2, M phases. This enabled direct observation of cell cycle progression under varying conditions. This figure provides a summary of the key data collected from two sets of experiments using unsynchronized and S-phase synchronized cell cultures. Pulsed electric field exposure in unsynchronized cells significantly increased the average length of G1, G2, and M phases, while the S phase remained unchanged. In synchronized cells, pulsed electric field exposure led to reduced durations of G1, S, and G2 phases, though only S and G2 were statistically significant, while M phase remained unaffected.