This article describes a method for isolating Schwann cells from mouse sciatic nerve fibers. The process involves enzymatic digestion, centrifugation, and magnetic separation techniques to obtain a pure Schwann cell population.
Take a suspension of cells and debris from enzymatically digested mouse sciatic nerve fibers.
Pass through a strainer, removing debris.
Centrifuge the filtrate and remove the supernatant.
Resuspend the cells in serum-containing media and seed them onto an adhesion protein-coated culture plate.
Incubate for cell adhesion. Replace media with Schwann cell media. Incubate for Schwann cell monolayer formation.
Remove the media and wash with buffer. Incubate with trypsin to detach cells.
Add media containing serum to stop the enzymatic activity.
Collect the cells and centrifuge. Discard the supernatant.
Resuspend the cells in buffer and add antibody-coated magnetic microbeads.
Incubate to bind antibodies to fibroblast glycoproteins.
Centrifuge and discard the supernatant. Resuspend the cells in buffer, and load them into a magnetic separation column containing ferromagnetic spheres placed in the magnetic field of the magnetic separator.
The magnetic field retains the microbead-bound fibroblasts, allowing Schwann cells to be collected in the flow-through.
Transfer the digested nerves into a 50-milliliter tube to centrifuge at 188 g for 10 minutes at 4 degrees Celsius. After discarding the supernatant, resuspend the pellet in 10 milliliters of DMEM containing 10% FCS and 50 micrograms per milliliter of gentamicin.
Next, filter the cell suspension through a 100-micrometer cell strainer. After centrifuging at 188 g for 10 minutes at 4 degrees Celsius, resuspend the pellet with 4 milliliters of DMEM containing FCS and gentamicin. Add 2 milliliters of the cell suspension to each of the two poly-L-lysine and laminin-coated 60-millimeter tissue culture dishes. Incubate at 37 degrees Celsius and 5% carbon dioxide, leaving the plates untouched for 2 days in the incubator.
Centrifuge the Schwann cell suspension at 188 g for 10 minutes at 4 degrees Celsius, and resuspend the cell pellet in 90 microliters of magnetic cell separation buffer per 1 x 107 cells. Then, add 10 microliters of Thy-1 microbeads per 1 x 107 cells. Incubate the resuspended solution for 15 minutes in the dark at 8 degrees Celsius.
Next, add 2 milliliters of the magnetic cell separation buffer to the cell suspension. After centrifuging at 300 g for 10 minutes at 4 degrees Celsius, resuspend the pellet in 500 microliters of magnetic cell separation buffer. Place the magnetic cell separation column in the cell separator, and moisten the column with 1 milliliter of the magnetic cell separation buffer. Then, apply the cells to it to collect the flow through.
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