Magnetic Isolation of Microglia from Mouse Pup Brains
Trascrizione
Prepare dissociation mixture according to this table. Transfer 12 brain pieces into a C-tube for a total weight of 1.2 grams per dissociation tube. Then, place C-tubes on the dissociator with heating. Start the optimized NTDK program in the dissociator. Centrifuge for 20 seconds. Complete the mechanical dissociation by pipetting three times.
Transfer the cells to four 15-milliliter tubes with attached strainers. Rinse the strainers with 10 milliliters of HBSS with calcium and magnesium. Centrifuge for 10 minutes, and remove the supernatant with a 10-milliliter pipette. Carefully, add 10 milliliters of HBSS with calcium and magnesium, and resuspend the pellet. Again, centrifuge and remove the supernatant.
Resuspend the pellet with 6 milliliters of sorting buffer. Repeat the centrifugation, and discard the supernatant. Then, add 200 microliters of CD11b-microbead solution, and incubate the tubes for 15 to 20 minutes at 4 degrees Celsius. After incubation, resuspend the pellet with 6 milliliters of sorting buffer. Repeat the centrifugation, and resuspend the pellet with 8 milliliters of sorting buffer.
Next, follow the POSSEL program on the separator to prepare eight columns. Pass the cells through the column by adding 1 milliliter of cell suspension at a time. With 1 milliliter of sorting buffer, elute CD11b positive cells on a sterile elution plate. Pool the cells in a 50-milliliter tube.
Centrifuge and resuspend the pellet with 10 milliliters of cold microglia medium. Count the CD11b positive cells. Resuspend the cells in cold microglia medium to get a final concentration of 650,000 to 700,000 cells per milliliter.
