Begin by placing the isolated mouse brain tissue in 1 milliliter of digestion buffer per brain in separate Petri dishes on ice. Thoroughly chop the brain tissues into small pieces using a clean scalpel blade. Using a plastic transfer pipette with its tip cutoff, carefully transfer minced brains into individual wells inside a 24-well plate on ice.
Cover the plate with a large piece of transparent, flexible film, close the lid, and incubate on ice for 30 minutes. Next, transfer the digested brain solution into individual 7-milliliter iced glass dounce homogenizers on ice containing 5 milliliters of cold FACS buffer. Gently dounce each brain with a loose pestle until a single-cell suspension is obtained.
Softly dounce with the tight pestle three to four times to ensure a single-cell suspension before transferring to 15-milliliter polypropylene tubes.
