Magnetic Separation of Schwann Cells from Mouse Sciatic Nerves
Transcrição
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.
