Isolating and Culturing Mouse Cerebral Pericytes
筆記録
Begin by placing the brain from a specific pathogen-free four to six-week-old male C57 black 6 mouse onto a sterile, dry, lint-free wipe and using curved tip forceps to remove the cerebellum, striatum, and occipital nerves. Use a cotton swab to remove all of the visible meninges, and turn the brain tissue upside down. Open the lobes with light outward strokes and remove all of the visible blood vessels. Then, place the meninges-free brain tissue in a 100-millimeter Petri dish containing 15 milliliters of cold Washing Buffer B.
To homogenize the tissue, transfer the brain sample into a Dounce Tissue Grinder mortar tube, and add 3 to 4 milliliters of Washing Buffer B to the tube. Use a loose pestle to mince the tissue 55 times. Then, rinse the pestle with Washing Buffer B, and mince the tissue slurry with a tight pestle 25 times. At the end of the homogenization, equally aliquot the slurry between two 50-milliliter tubes and add 1.5 times the volume of cold 30% bovine serum albumin dextran. Then, vigorously shake the tubes to mix the slurry.
To isolate the vascular fraction, sediment the cells by centrifugation, and transfer the supernatants into two new tubes. Store the pellet in Washing Buffer B on ice. Resuspend the pellets in three milliliters of cold Washing Buffer B on ice.
Centrifuge the harvested supernatants, and repeat this step further one more time. Discard the supernatants, and resuspend the pellets in 3 milliliters of Washing Buffer B. Then, pool the resuspended pellets and bring the final volume of the pooled cell suspensions to 10 milliliters with fresh cold Washing Buffer B.
Further, dissociate the cells with a 10-millimeter pipette six times until no remaining clumps of pellets are visible, and use a vacuum filter assembly and a nylon mesh strainer to filter the cell suspension. Place the strainer in a Petri dish, and clear the mesh with fresh Washing Buffer B and scraping to recover any capillaries. Then, perform a second filtration with a fresh filter.
Divide the filtrate equally between two tubes and collect the cells by centrifugation. After discarding the supernatants, pool the pellets into a single tube containing pre-warmed Washing Buffer B with enzymes and add pre-warmed collagenase dispase to the tube. Place the tube in a shaking table water bath for precisely 33 minutes at 37 degrees Celsius before stopping the reaction with 30 milliliters of cold Washing Buffer B.
Collect the cells by centrifugation, and carefully discard the supernatant. Quickly but carefully resuspend the pellet in Washing Buffer B six times and centrifuge the suspension again. After discarding the supernatant, resuspend the pellet in milliliters of complete DMEM medium and seed 2 milliliters of cell suspension in complete DMEM in nine wells of three Matrigel-coated six-well plates.
Place the cell cultures in a sterile 37 degrees Celsius, 5% carbon dioxide incubator for 24 hours before carefully removing the debris from each well, and add fresh DMEM media. After 48 hours, change the medium in each well every 48 hours. On day 8 to 10, when the cells reach 100% confluency, passage the cells in parasite culture medium onto new gelatin-coated six-well plates and return the cells to the cell culture incubator for an additional 6 to 7 days with monitoring.
