To begin, isolate and culture the outer layer of the aorta and the first branch of the mesenteric arteries isolated from the anesthetized mouse. Once the cells become 80%confluent, centrifuge the dissociated cell suspension at 300G for five minutes at room temperature, discard the supernatant completely and resuspend the cell pellet in 98 microliters of sorting buffer per 10 million total cells. After the addition of CD34 antibody, incubate the cells in the dark for 30 minutes at four degrees Celsius.
To wash the cells, add two milliliters of sorting buffer to the cells, and centrifuge at 300G for 10 minutes. After discarding the supernatant, resuspend the cells in 80 microliters of sorting buffer. Then add 20 microliters of Anti-FITC MicroBeads to the cell suspension.
After washing and centrifuging the cells as demonstrated previously, resuspend the cells in one milliliter of sorting buffer. Next, position a magnetic separation column within the magnetic field of a separator, and place a collection tube beneath the column. After rinsing with 500 microliters of buffer, load the cell suspension into the column and collect the flow through containing unlabeled cells in the collection tube.
Remove the column from the separator and place it on a fresh 15 milliliter centrifuge tube. Introduce 500 microliters of sorting buffer into the column and push the plunger into the column to flush out the magnetically labeled cells.
