Purifying Capillaries From a Porcine Brain
Trascrizione
To begin this procedure, place the brains in a sterile flow bench, and gently wash them with 1 liter of PBS in a beaker placed on ice. Next, carefully remove the meninges from each brain using fine-tip forceps. Using a scalpel, scrape off gray matter from the brains one at a time, and transfer the isolated material to Petri dishes containing 20 milliliters of DMEM/F12 placed on ice.
For initial fragmentation, run the gray matter material through a 50-milliliter syringe without the needle. Repeat the procedure for all brains and pool the collected gray matter material together. Transfer the isolated gray matter material with DMEM/F12 medium to the grinder tube of a handheld tissue homogenizer. Homogenize the material by making 8 up and down strokes with a loose pestle, followed by 8 up and down strokes with a tight pestle.
Next, isolate the capillaries by filtering the homogenate using a 500-milliliter blue cap bottle with filter holder and 140 micron-filter mesh. Place the capillary containing filters in Petri dishes with the digestion solution. Subsequently, incubate the Petri dishes at 37 degrees Celsius for one hour on an orbital shaker at 180 RPM, or stir them gently every 10 minutes.
After one hour, wash off the capillaries from the filters with suspension from the Petri dish. Then, split the suspension from the three dishes into two 50-milliliter tubes, and stop the digestion by adding 10 milliliters of DMEM/F12 to each 50-milliliter tube.
Centrifuge the cell suspensions at 250 times g, 4 degrees Celsius for 5 minutes. Afterward, aspirate the supernatants, and resuspend each pellet in 10 milliliters of DMEM/F12. Then, add a further 20 milliliters of DMEM/F12 to each tube.
