Our research focuses on promoting peripheral nerve regeneration by nsPEF stimulated Schwann cells. We hope to develop a new way to activate Schwann cells cells efficiently as nerve regeneration is quite difficult in most cases. We innovatively use a nanosecond pulsed electric field, nsPEF to activate Schwann cells.
We are delighted to find that nsPEF may be a preferred mode for peripheral nerve regeneration with minimal stimulation side effects. Besides our protocol provide a reliable basis for subsequent in depth and systematic research. Our laboratory will continually explore the application of nsPEF.
In terms of nerve regeneration, we'll build up animal models to promote in vitro testing and evaluate the safety of nsPEF in vitro. As there is limited evidence regarding the application of nsPEF in peripheral nerve regeneration, we hope we can make breakthroughs. To begin place the cryo vial containing one milliliter of RSC96 cell suspension in a 37 degrees Celsius water bath while rapidly shaking it.
Transfer the cell suspension to a centrifuge tube containing four to six milliliters of complete culture medium and mix well. Centrifuge the tube at 1000 G for three to five minutes, then discard the supernatant and resuspend the cells in three milliliters of complete culture medium. Now add the cell suspension to a culture flask containing six to eight milliliters of complete culture medium and incubate at 37 degrees Celsius overnight.
Once the cell density reaches 80 to 90%discard the culture medium and rinse the cells one to two times with PBS without calcium and magnesium ions. Then add 0.25%trypsin, 0.53 millimolar EDTA to the culture flask. At the end of the incubation, observe the cell detachment under a microscope.
Once most cells become round and detached quickly return the flask to the working area and tap it gently. Add three to four milliliters of culture medium containing 10%FBS to stop the digestion. Then mix the contents of the flask thoroughly and aspirate the solution.
Centrifuge the cell suspension. Add 1000 G for five minutes. After discarding the supernatant, resuspend the cells in one to two milliliters of fresh culture medium with gentle pipetting.
Transfer the cell suspension to a new T25 flask at a one to two ratio and add seven milliliters of culture medium. To begin resuspend the cultured RSC 96 cells in one milliliter of DMEM culture medium and transfer them to colorimetric dishes with electrodes on both sides. Turn on the power switch of the instrument and rotate the knob to set the intensities of the electric field.
Carefully rotate the electrodes until sparks appear. Allowing the cells to receive five pulses of nanosecond pulsed electric fields according to the preset field strength intensities. Immediately separate the two electrodes after the treatment.
To begin, take the electrically stimulated RSC96 cells and add 100 microliters of the cells to each well of a 96 well cell culture plate. Take 10 microliters of CCK-8 solution from the kit and add it to the 96 well cell culture plate. Incubate the plate in a carbon dioxide incubator at 37 degrees Celsius for an additional 30 minutes to four hours.
Seed three times 10 to the fifth cells in each well of a six well plate with a total volume of two milliliters per well. Use a pipette tip to draw a horizontal line at the bottom of the culture well while holding the tip vertically. After removing the culture media, wash the cells with PBS two to three times.
Add two milliliters of serum free medium to each well then place the plate in the 37 degrees Celsius incubator. Take pictures under a fourfold magnification of the inverted microscope at zero hours and 24 hours to observe changes in cell migration. The CCK-8 assay revealed that RSC96 cells proliferated significantly faster in the five kilovolts per centimeter group compared to the control.
In contrast, higher parameters led to unstable proliferation rates than the control and five kilovolts per centimeter groups. The scratch assay revealed that the migration rate of RSC96 cells in the five kilovolts per centimeter group was significantly faster than that of the control and 10 kilovolts per centimeter groups. To begin, take electrically stimulated RSC96 cells in culture dishes.
Then use a histology pen to draw circles at locations where cells are evenly distributed on the cover slip. Add 50 to 100 microliters of permeabilization working solution to the cells and incubate at room temperature for 20 minutes. Then wash the cells three times with PBS for five minutes each.
Next, add 3%BSA within the circles to cover the tissue uniformly and incubate at room temperature for 30 minutes. Afterwards, gently remove the blocking solution and add the appropriately diluted primary antibody to the cells. Place the cell culture plate in a humid box and incubate overnight at four degrees Celsius.
Then wash the cells three times with PBS for five minutes each while shaking continuously. Add the corresponding secondary antibody and incubate at room temperature for 50 minutes. At the end of the incubation, place the cover slip in PBS and wash it three times while shaking for five minutes each.
Then allow the slide to dry and add DAPI staining solution to the dried slide. Finally, seal the dried cover slip with an anti fading mounting medium for fluorescence. Acquire images using specific excitation and emission wavelengths.
For Alexa Fluor 488 SCI 3 and SCI 5. Microscopic analysis showed scattered cytoplasmic S100-Beta positive cells in red across all groups. An increased integrated optical density of S100-Beta fluorescence was observed in the five kilovolts per centimeter group compared to the control and 10 kilovolts per centimeter groups.
GFAP and Sox10 positive cells with scattered cytoplasm were observed in all groups during cell crawling assays. However, GFAP expression was significantly lower in the five kilovolts per centimeter group compared to the control and 10 kilovolts per centimeter groups. RSC96 cells in the five kilovolts per centimeter group showed a significantly higher mean gray value of Sox10 expression suggesting enhanced Sox10 expression.
