Processing Murine Small Intestine for Crypt Isolation

Begin by isolating the small intestine from the euthanized animal and transfer the tissue to a Petri dish. Using laboratory scissors and tweezers, remove the fat tissues from the small intestine. Then, cut the small intestine into several segments.

Using a five-milliliter syringe, flush the small intestine with five milliliters of cold PBS-antibiotics to clear the luminal content. Cut the tissue open lengthwise using laboratory scissors. Then, manually wash it with cold PBS-antibiotics while shaking.

Using laboratory scissors, cut the intestinal segment into pieces measuring approximately five millimeters by five millimeters. Use a pipette to transfer the collected fragments into a 50-milliliter tube. Next, add 25 milliliters of cold PBS-antibiotics to the tube containing the tissue fragments and shake the tube back and forth 10 times to wash off the intestinal contents from the tissue fragments.

Aspirate the buffer after washing. The tissue fragments are now ready for crypt isolation.