Intestinal Crypts Isolation: A Method to Isolate Whole Crypts from Small Intestine of Murine Model
Trascrizione
Dissect the peritoneum longitudinally with a pair of scissors. Hold the stomach with the forceps and cut it transversely in half using intestinal scissors from this point forward. Pull out the intestine and place it in a Petri dish containing PBS. Start by placing the blunt tip into the stomach and gently push it through the pylorus.
Proceed by cutting with the scissors and pulling with the forceps. Once the whole small intestine is open longitudinally, wash it in cold PBS by holding it with the forceps and gently rinsing it in the PBS solution with U-shaped movements. Once all the stool remnants are cleared, proceed to flatten the intestine on a cutting board luminal side up.
The luminal side is easily recognizable by the absence of blood vessels and by its pale appearance when compared with the outer part. With a glass slide, gently removed the villa by scraping the flattened intestine. Perform this step twice along the whole length of the tissue. Then, cut the small intestine with a sterile surgical blade into 2- to 5-millimeter pieces.
Place the small intestinal fragments in a 50-milliliter tube containing 10 milliliters of ice-cold PBS. Clean the tissue fragments removing any remaining impurities by pipetting them up and down in the PBS. Discard the supernatant and repeat this step until the PBS is completely clear. Next, add 15 milliliters of cold PBS to bring the total final volume of PBS to 25 milliliters. Add 2 millimolar EDTA and incubate for 45 minutes on a roller at 4 degrees Celsius.
After discarding the PBS/EDTA, add 10 milliliters of PBS and detach the crypts by harshly pipetting the tissue fragments up and down. Then, collect the supernatant. Repeat the step four times. Add culture medium to reach a final volume of 50 milliliters. Pellet the cells by centrifugation. Finally, resuspend the pellet in 10 milliliters of culture medium before pelleting the crypts by centrifugation.
