Isolating Murine Primary Oligodendrocytes Using Immunomagnetic Beads

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.