Intestinal Crypts Isolation: A Method to Isolate Whole Crypts from Small Intestine of Murine Model
Transcrição
The intestinal lining is covered with projections or villi and invaginations or crypts. Crypts are the intestinal glands that primarily secrete digestive enzymes.
To isolate small intestinal crypts, begin by positioning an anesthetized mouse in the supine position on a dissection board. Using scissors, dissect the peritoneum longitudinally to expose the digestive system.
Locate the small intestine and gently pull it up from the mouse's body. Snip the intestine at its base. Transfer the harvested intestine to a dish containing a suitable buffer. Cut through the intestine longitudinally, exposing the lumen.
Place the intestine, luminal side up, on a cutting board to expose the villi. Move an angled glass slide over the intestinal surface to scrape off the villi, exposing the whole crypts.
Cut the intestine into tiny pieces, and transfer the pieces into a tube containing an ice-cold buffer to ensure minimal damage to the crypts.
Next, treat with EDTA and incubate. EDTA causes the whole crypts to loosen from the intestinal lining. Remove the spent EDTA-containing solution. Add fresh buffer and triturate using a pipette to detach the crypts from the intestinal lining.
Collect the supernatant containing the crypts into a fresh tube. Supplement the tube with a suitable culture medium and centrifuge to pellet the crypts.
