Magnetic Separation of Schwann Cells from Mouse Sciatic Nerves
Transcrição
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.
