Isolating Adipose-Derived Stem Cells from Murine Periaortic Adipose Tissue
Transcription
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.
