Neuronal Enrichment of Dorsal Root Ganglia by Immunopanning
Transcription
Begin by layering the dorsal root ganglia-derived cells onto bovine serum albumin or BSA.
The cell suspension contains neurons, immune cells, fibroblasts, Schwann cells, and fatty myelin debris.
Centrifuge. The denser DRG cells pass through, forming a pellet.
Remove the myelin layer along with the serum.
Resuspend the cells in an immunopanning buffer, which improves the accessibility of the antigen for antibodies.
Take Petri dishes coated with secondary antibodies conjugated with cell-specific primary antibodies.
Add the cell suspension to the immune cell-specific antibody-coated Petri dish.
The immune cells attach to the antibodies while the other cells remain unbound.
Transfer the remaining cells to the next Petri dish to remove the fibroblasts.
Repeat the procedure to remove the Schwann cells.
Plate the unbound cells onto a matrix-coated multiwell plate containing a neurobasal medium and incubate to obtain an enriched neuronal culture.
