Encyclopedia of Experiments: Immunology
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Transcript
To begin, resuspend the CD34-positive cell pellet isolated from cord blood in 300 microliters of magnetic cell separator buffer per 1 x 108 cells. Add 100 microliters of FcR blocking reagent per 1 x 108 cells, followed by 100 microliters of CD34-positive magnetic beads per 1 x 108 cells, and incubate at 4 degrees Celsius for 30 minutes.
Next, add 5 milliliters of magnetic cell separator buffer per 1 x 108 cells and spin at 360 g for 10 minutes at 4 degrees Celsius. Repeat the wash step and resuspend the pellet in 500 microliters per 100 million cells of magnetic cell separator buffer in a 15-milliliter conical tube labeled 'unfractionated.' Label two more 15-milliliter tubes—CD34-negative and CD34-positive. Place the three tubes on a cooling rack.
Separate the cells using the two-column positive selection program on an automatic magnetic cell separator in a biosafety cabinet. Next, for expanding and freezing CD34-positive human stem cells, prepare cord blood or CB medium as described in the manuscript and pass through a 0.22-micron filter. Resuspend the CD34-positive cells at 100,000 per milliliter of CB medium, and incubate at 37 degrees Celsius.
On day three, add an equal volume of CB medium without cytokines to the flask. On day five, harvest the expanded CD34-positive cells. Pipette the cell suspension up and down and collect in a 50-milliliter conical tube. Add enough CB medium to cover the bottom of the flask. Using a cell scraper, scrape the entire bottom of the flask. Collect all the media into the same 50-milliliter tube, and centrifuge.
Resuspend the cells in 2 milliliters of CB medium, and centrifuge. Aspirate the medium down to the pellet, and resuspend the pellet in a freezing medium. Next, begin CD34-positive cell preparation three hours post-pup irradiation. Warm 10 milliliters of CB media in a 50-milliliter tube.
Retrieve one vial of in vitro expanded and frozen CD34-positive cells for every four to six pups to inject. Rapidly thaw at 50 to 55 degrees Celsius, until just a small amount of ice is visible, and add the cells to the warmed CB medium. Use 1 milliliter of medium to rinse each vial and spin the cells at 360 g for 12 minutes at 4 degrees Celsius.
Aspirate the medium carefully. Resuspend the pellet in 2 milliliters of CB media and count the cells. Again, centrifuge the cells, aspirate the medium, and resuspend the pellet in 100 microliters of sterile PBS per n plus 1 pups to inject, resulting in 250,000 to 450,000 CD34-positive cells per mouse.
