This article describes a method for isolating primary oligodendrocytes from a neural cell suspension derived from mouse pups. The process involves the use of engineered magnetic microbeads to selectively bind to oligodendrocytes, facilitating their separation from other cell types.
Take a neural cell suspension, including immature oligodendrocytes and primary neuronal cells, from a mouse pup.
Resuspend the cells in a magnetic cell sorting buffer to avoid cell clumping.
Add engineered magnetic microbeads carrying anti-O4 antibodies on their surface. Agitate the solution to ensure uniform mixing.
Incubate to enable microbeads to bind with sulfated galactolipid, an O4 antigen on oligodendrocytes, while other cells remain unbound or loosely bound.
Wash the mix with additional cell sorting buffer.
Centrifuge and discard the supernatant, then resuspend the cells.
Next, pass the bead-bound cells into a magnetic separation column equipped with a filter that removes the larger cell clumps.
During the run, under a magnetic field, the bead-bound complexes move toward the magnet, attaching to the sides.
Wash to remove unbound and loosely bound cells.
Retrieve the column from the separator and wash it with a proliferation medium to collect the primary oligodendrocytes for future applications.
After counting, centrifuge the cells again, and resuspend the pellet in 90 microliters of fresh magnetic cell-sorting buffer per 1 x 107 cells. Next, add 10 microliters of anti-O4 beads per 1 x 107 cells, and mix the cell solution with gentle flicking. After 15 minutes at 4 degrees Celsius, with gentle flicking, every 5 minutes, gently add 2 milliliters of magnetic cell-sorting buffer per 1 x 107 cells for a wash by centrifugation, and discard the supernatant by careful vacuum aspiration.
Resuspend the pellet in 500 microliters of fresh magnetic cell-sorting buffer for every 1 x 107 cells and place an appropriately-sized magnetic bead-sorting column into its corresponding magnetic separator. Place a 40-micron strainer onto the column and pre-rinse the strainer with 3 milliliters of magnetic cell-sorting buffer, letting the buffer run through the column without letting the column dry. Add the cells to the strainer, followed by a 1-milliliter wash with magnetic cell-sorting buffer.
Wash the column three times with 3 milliliters of magnetic cell-sorting buffer per wash, and one time with oligodendrocyte proliferation medium. Then, transfer the column into a 15-milliliter tube and use the plunger to immediately flush 5 milliliters of fresh oligodendrocyte proliferation medium through the column.
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