This article details a protocol for isolating fluorescent protein-expressing adipose-derived stem cells from transgenic mouse periaortic adipose tissues. The process involves tissue digestion, cell sorting, and culture setup for further experimentation.
Take harvested transgenic mouse periaortic adipose tissues containing fluorescent protein-expressing adipose-derived stem cells descended from neural crest cells, or NCCs.
Add digestion media and mince the tissues.
Transfer the minced tissue with the digestion media to a tube. Mechanically dissociate the fragments.
Incubate. Collagenase in the media digests the tissue's extracellular matrix, releasing stem cells, adipocytes, and fibroblasts.
Add serum-containing media to stop collagenase activity. Centrifuge and discard the supernatant containing adipocytes.
Resuspend the cells in media, and filter to remove aggregates.
Centrifuge the filtrate and remove the supernatant. Resuspend the cells in erythrocyte lysis buffer to lyse erythrocytes.
Add serum-containing buffer to stop the lysis. Centrifuge and discard the supernatant.
Resuspend the cells in buffer.
Perform fluorescence-activated cell sorting to isolate fluorescent stem cells.
The isolated stem cells can be used for further experiments.
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.
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