Isolating Adipose-Derived Stem Cells from Murine Periaortic Adipose Tissue
Transcript
Transfer the harvested adipose tissue into a new 2-milliliter microcentrifuge tube, containing 1 milliliter of freshly prepared digestion medium, and use surgical scissors to mince the tissues. Transfer the tissue slurry into a 50-milliliter tube containing 9 milliliters of fresh digestion medium, and use a 1-milliliter pipette to triturate the tissues 10 times.
When a homogeneous solution has been obtained, incubate the tube for 30 to 45 minutes at 37 degrees Celsius and 100 revolutions per minute, checking every 5 to 10 minutes to prevent over-digestion. When a light yellow homogeneous adipose tissue solution can be observed upon gentle swirling of the tube, stop the digestion with 5 milliliters of HDMEM supplemented with 10% fetal bovine serum. After a thorough mixing, centrifuge the adipose tissue sample.
The stromal vascular cell fraction will be visible as a brownish pellet. Resuspend the pellet in 10 milliliters of culture medium, and filter the cell suspension through a 70-micrometer cell strainer.
Collect the cells by another centrifugation, and gently resuspend the pellet in 5 milliliters of erythrocyte lysis buffer. Transfer the suspension to a 15-millimeter tube. After 10 minutes, stop the reaction with two centrifugations in 10 milliliters of PBS, supplemented with 1% fetal bovine serum.
After the second centrifugation, resuspend the pellet in 5 milliliters of culture medium on ice, and collect the cells with a final centrifugation. Then, resuspend the cells in 5 milliliters of FACS buffer for counting.
To set up an NCADSC culture after their isolation by FACS according to standard protocols, place the cells at a 5 x 103 cells per cm2 concentration in each well, a 12-well culture plate in complete culture medium, at 37 degrees Celsius, and 5% carbon dioxide for 20 to 24 hours. The next day, wash the cells with 37 Celsius PBS, and feed the culture with fresh culture medium.
