Isolation in phenotypic analysis of immune cells from the intestine using this protocol have proven applicable to the understanding of GI and systemic inflammatory disorders. This method isolates a significant number of viable mononuclear cells by minimizing contaminating debris allowing subsequent immune phenotyping by Fleur's Atomistry or other methods. As the intestine is a major target site of inflammation in many diseases, this protocol may be useful for the study of intestinal immune populations in mouse models of cancer, allergy, transplantation, and autoimmunity.
To optimize the yield and efficiency of this protocol, it is recommended that key solutions and materials be prepared in advance as noted. After euthanizing the mouse and making a vertical midline incision, use fine dissection scissors to open the peritoneum. Using forceps, move the small bowel to one side and expose the descending colon.
Pull upwards slightly on the descending colon to maximally expose the rectal portion of the colon. Then, cut the distal rectum deep in the pelvis and dissect the entire colon as one unit from the distal rectum to the cecal cap. First, place the colon on a moistened paper towel and use the blunt end of a pair of scissors or forceps to apply mild pressure to the bowel wall and extract the solid stool.
Next, place the colon in a petri dish and use a 10 milliliter syringe with an 18-gauge blunt filled needle to flush the gut with 10 milliliters of chilled colon buffer. Place the colon in a petri dish filled with 5-10 milliliters of chilled colon buffer and agitate manually to wash the remaining colonic contents. Repeat this wash 2-3 times, using a new petri dish containing fresh buffer for each wash.
After this, transfer the colon to a new petri dish containing fresh chilled colon buffer. Cut the colon longitudinally from its more muscular rectal end to the proximal colon, to generate a single rectangular open colon piece. Discard the median in the dish and replace it with clean chilled colon buffer.
Place the rectangular colon tissue on a paper towel that is moistened with colon buffer and cut it by slicing it horizontally and then into small fragments. Use fine forceps to collect the colon fragments into a 50 milliliter polypropylene conical tube containing 20 milliliters of chilled colon buffer. Then, wash the fragments by vigorously swirling the tube for 30 seconds.
After the wash, let the tissue fragments settle to the bottom of the tube before decanting or vacuum aspirating the supernatant, Making sure to avoid losing tissue fragments, repeat this entire wash process 3 times using fresh colon buffer for each wash. To begin, add 20 milliliters of collagenase digestion buffer to the tube containing the washed colon fragments. Seal the tube and place it in an orbital shaker at 37 degrees Celsius with a rotation rate of 2 times G for 60 minutes.
Ensure the tissue fragments are in constant motion during agitation. First, pour 5 milliliters of 66%silica based density separation media into each of 3 separate 15 milliliter polypropylene tubes. Next, use a 25 milliliter serological pipette to collect only the supernatant from Collagenase Digestion 1.
Filter the supernatant through a 40 microliter poor filtration fabric cell strainer placed into a clean 50 milliliter polypropylene conical tube. Fill the tube completely with chilled colon buffer to quench the collagenase digestion buffer. Then spin the cells, aspirate the supernatant, and keep the pellet on ice.
Continue to perform Collagenase Digestion 2 as outlined in the text protocol. After Collagenase Digestion 2 is completed, flush the tissue fragments vigorously back and forth between the tube and a 10 milliliter syringe through an 18-gauge blunt end needle. Repeat this flush for at least 7-8 passages, continuing until no gross tissue fragments or debri are visible.
Next, pass the tissue disaggregation suspension through a 40 micrometer poor filtration fabric cell strainer placed into a clean 50 milliliter polypropylene tube. Fill the tube to the rim with chilled colon buffer to quench the digestion and centrifuge at 800 times G in at 4 degrees Celsius for 5 minutes. Discard the supernatant via vacuum aspiration and pull the re suspended pellet from Collagenase Digestion 1 to its corresponding tube.
After this, re suspend each pellet in 24 milliliters of 44%silica based density separation media per colon. Use a 10 milliliter serological pipette to slowly layer 8 milliliters of this media onto each of 3 tubes containing 66%silica based density separation media. Carefully balance the tubes within centrifuge buckets using a weigh scale or a balance.
Centrifuge at 859 times G in at 20 degrees Celsius for 20 minutes without the break. Allow the roders to come to complete rest before removing tubes, taking care not to disrupt the cells at the gradient interface. First, visualize the gradient interface near the 5 milliliter mark, where typically a 1-2 milliliter thick white band is present.
Vacuum aspirate and discard the top 7 milliliters of the gradient to allow easier pipette access to the interface. Using continuous manual suction and steady rotating wrist motion, collect the interface layer of cells. Collect until the interface between the two gradients is clear and refractile and transfer the interface to a new 50 milliliter polypropylene conical tube.
Next, add FACS Buffer to this tub until the total suspension reaches 50 milliliters. Centrifuge at 800 times G in at 4 degrees Celsius for 5 minutes. Then, aspirate the supernatant via vacuum aspiration and re suspend the pellet in 1 milliliter of FACS Buffer.
The procedure described in this video can be used to isolate mononuclear cells from the colon or, with modifications, from the small intestine. Plus, cytometry and data analysis is performed to compare the fractions of apoptotic and necrotic/dead lymphocytes when using either Collagenase E or D for the isolation, with our without DNAse 1 treatment. Following Annexin 5 and fixable live dead blue dye staining on single cell suspensions following each isolation, Collagenase E without DNAse showed a significantly higher percentage of live cells after isolation when compared to Collagenase D without DNAse.
Even when compared to either collagenase with DNAse. In addition, we identified necrotic cells at a median percentage of 41.0%in the Collagenase E group versus 90.0%in the Collagenase D group. 75.9%in the Collagenase E DNAse group and 80.3%in the Collagenase D DNAse group.
Multi perimetric flow cytometry is then applied to MNC isolated from CD45.2 valve-C recipient mice on Day 7 after receiving BMT or either allogeneic or syngeneic BMT models. The mean absolute numbers of Donor CD4 positive and CD8 positive T-cells extracted from the BMT recipient's colon are calculated and compared. So it can be important to identify and/or quantitate rare immune cell populations in such mouse models.
Rare subsets, including donor derived FOX-P3 positive T regulatory cells are assessed in both syngeneic and allogeneic BMT models. Using this method, even rare subsets, such as donor derived colonic T regulatory infiltrating recipient mouse colon, after BMT, can be analyzed. It should be noted that Collagenase E is active at 37 degrees Celsius.
So all collagenase solutions should be pre-warmed for adequate collagenase activity and thoroughly quenched to terminate activity. This protocol allows for a large number of mononuclear cells that can be used for subsequent characterization by a Fleur cytometry, molecular biology techniques, microscopy, culture, and site of, among others. This protocol provided a dependable assessment of inflammation in the colon of experimental versus controlled mice in a bone marrow transplant model.
Thus facilitating the discovery of novel regulatory immune mechanisms of transplant tolerance. Collagenase from Clostridium Istoliticum is a reagent that should be handled with lab safety techniques. It poses a health hazard when inhaled and may cause skin and eye irritation.
