Isolation of Schwann Cells from Mouse Spinal Roots
Transcrição
Slice the nerves into 3- to 5-millimeter pieces with scissors. Add 1 milliliter of 0.25% trypsin, and transfer the nerve pieces to 5-milliliter centrifuge tubes. Incubate at 37 degrees Celsius for 18 to 20 minutes. Next, to stop the digestion, add 3 to 4 milliliters of DMEM containing 10% fetal bovine serum. Pipette the mixture up and down gently, about 10 times. Centrifuge at 800 times g for 5 minutes.
After centrifugation, discard the supernatant, and resuspend the precipitate in 2 to 3 milliliters of DMEM supplemented with 10% FBS. Filter the cell suspension through a 400-mesh filter, and then use the suspension to inoculate a 60-millimeter Petri dish. Incubate the Petri dishes for 4 to 5 days at 37 degrees Celsius in the presence of 5% carbon dioxide.
When the culture has reached 90% confluence, wash the cells once using 1X PBS. Then, to digest the Schwann cells, add 1 milliliter of 0.25% trypsin at 37 degrees Celsius per 60-millimeter dish. After 8 to 10 seconds at room temperature, stop the digestion by adding 3 milliliters of DMEM supplemented with 10% FBS. To detach the Schwann cells, gently blow on the cells with a pipette.
Verify by microscope that the cells are detached. Then, collect the medium, which contains the Schwann cells, and centrifuge at 800 x g for 5 minutes. Discard the supernatant, and resuspend the precipitate in 3 milliliters of medium. Use the suspension to inoculate uncoded 60-millimeter Petri dishes, and incubate the dishes at 37 degrees Celsius for 30 to 45 minutes. Transfer the medium, which will contain the Schwann cells to a poly-L-lysine-coated medium dish, and incubate the dish at 37 degrees Celsius for 2 days.
