The overall goal of this protocol is to isolate lymphocytes from intestinal inductive sites including Peyer's patches and mesenteric lymph nodes and effector sites including lamina propria and epithelium. This method can help answer key questions in the mucosal immunology field such as understanding the immune responses to gastrointestinal infections, cancers, and inflammatory diseases. The main advantage of this technique is that it ensures the isolation of highly purified and viable lymphocytes from distinct compartments of the small intestine with minimal cross-compartmental contamination.
Generally, individuals new to this method will struggle because it is challenging to identify and isolate mesentery lymph nodes and Peyer's patches and to balance speed and precision for optimal yields. To begin the protocol, place a previously anesthetized mouse on its back and spray it with 70%ethanol. Use scissors to make a midline incision and open the skin and abdominal wall to expose the peritoneal cavity.
Next locate the cecum and use forceps to gently pull down the cecum. Use forceps to carefully move the small intestine to the right exposing the entire Mesenteric Lymph Nodes or MLN chain which is aligned with the colon. Identify the bottom node in the chain located closest to the cecum.
Use one set of forceps to grasp the mesentery fat around it and gently move the MLN chain from the bottom to the top by tweezing the fat around the lymph node with another set of forceps. Then moisten a towel with harvest media and lay the MLN chain on the paper towel. Remove mesentery fat by rolling the chain on the paper towel and pulling the fat off with two sets of forceps.
Place the MLN in cold harvest media for later isolation steps. With scissors, cut the small intestine below the pyloric sphincter and above the cecum. Pull the intestine out slowly from the ileum to duodenum using one set of forceps while removing the attached mesentery fat using another set of forceps.
Place the intestine on a paper towel moistened with harvest media and tease any remaining mesentery fat off with two sets of forceps. Keep the intestine moistened throughout these steps by applying harvest media periodically. Collect Peyer's patches by removing them from the intestine with curved scissors.
Collect 5-11 Peyer's patches from the small intestine. Place the Peyer's patches in cold harvest media for later isolation steps. Use the flat side of forceps and gently slide from the duodenum toward the ileum to expel fecal content and mucus.
Repeat this step once to remove most of the mucus. Use fine scissors with a blunt end on one point, insert the blunt end into the intestine and cut the intestine open longitudinally from duodenum to ileum. Laterally cut the opened intestine into two centimeter pieces.
Place the intestinal pieces in a 50 milliliter conical tube with 25 milliliters cold harvest media for later isolation steps. Isolate lymphocytes from the MLN by gently dissociating them through a 70 micrometer cell strainer using the plunger of a three milliliter syringe. Wash the cell strainer with five milliliters of cold harvest media.
Pellet the MLN cells by centrifuging them at 400 times gravity for five minutes at four degrees Celsius. Then resuspend the cell pellet in one milliliter cold harvest media. Wash the pieces of small intestine three times with 25 milliliters of cold harvest media by inverting the tube 10 times.
Allow the intestinal pieces to settle and pour off the supernatant. Next add 20 milliliters of prewarmed dithioerythritol or DTE solution to the 50 milliliter conical tube containing intestine pieces and transfer the contents to a 50 milliliter siliconized Erlenmeyer flask with a stir bar. Use a large magnet on the outside of the flask to hold the stir bar and transfer the tissue and solution back to the 50 milliliter conical tube.
Then vortex the tube at the maximum setting for 10 seconds. Transfer the supernatant into a new 50 milliliter conical tube through a 70 micrometer cell strainer, being careful to keep the tissue pieces in the original tube. Pellet the cells.
Resuspend the cell pellet in 10 milliliters cold harvest media and store on ice. Add 20 milliliters of prewarmed DTE solution to the 50 milliliter conical tube containing intestine pieces and transfer back to the 50 milliliter siliconized Erlenmeyer flask containing a stir bar. Use a large magnet on the outside of the flask to hold the stir bar and transfer the tissue and solution back to the 50 milliliter conical tube.
Vortex the tube at the maximum setting for 10 seconds. Transfer the supernatant through a 70 micrometer cell strainer into the tube containing cells from the supernatant of the first DTE treatment and centrifuge the cells. Resuspend the cell pellet in eight milliliters of 44%Density Gradient or DG solution at room temperature.
Transfer the eight milliliter 44%DG solution and cell suspension into a 17 millimeter by 100 millimeter 14 milliliter polypropylene round bottom tube. Underlay the cells with five milliliters of RT 67%DG solution. Centrifuge at 1, 600 times gravity for 25 minutes at RT without using the brake.
Viable cells form a buffy coat at the 44%and 67%interface. Remove the fat layer on top by vacuum aspiration. Use a Pasteur pipette to harvest the buffy coat at the interface and transfer to a 50 milliliter conical tube containing 40 milliliters cold harvest media.
Pellet cells by centrifuging at 400 times gravity for five minutes at four degrees Celsius and resuspend the cell pellet in one milliliter cold harvest media. Remove epithelial cells from intestinal tissue pieces by adding 25 milliliters of prewarmed EDTA solution to the remaining intestinal pieces and transfer the contents to a 50 milliliter siliconized Erlenmeyer flask with a stir bar and stir. Hold the stir bar and carefully discard the supernatant while keeping the intestinal pieces in the flask.
Repeat the epithelial cell removal step one more time. Next add 50 milliliters of room temperature harvest media and stir the contents briefly with a magnet. Then let the intestinal pieces settle and carefully discard the supernatant.
Add 30 milliliters of prewarmed collagenase solution and stir the intestinal pieces. After stirring, transfer the digested tissue and supernatant to a 50 milliliter conical tube through a 70 micrometer cell strainer. Gently dissociate the remaining tissue pieces by using the plunger of a three milliliter syringe to mash through the filter.
Wash the filter with 10 milliliters of cold harvest media and pellet the cells. After spinning, resuspend the cell pellet in eight milliliters of ambient temperature 44%DG solution. Transfer the eight milliliters of 44%DG solution cell suspension into a fresh 70 millimeter by 100 millimeter polypropylene round bottom tube.
Underlay with five milliliters of ambient temperature 67%DG solution then centrifuge the gradients. Remove the fat layer on top by vacuum aspiration. Use a Pasteur pipette to harvest the buffy coat at the interface and transfer to a 50 milliliter conical tube containing 40 milliliters cold harvest media.
Finally, resuspend the cell pellet in one milliliter of cold harvest media. Each intestinal immune compartment is typified by a unique composition of lymphocyte subsets. The MLN is predominantly composed of alpha beta T cells while PP lymphocytes are mostly B cells.
LP lymphocyte composition is primarily composed of alpha beta T cells and B cells. IEL are mainly gamma delta T cells and alpha beta T cells with essentially no B cells. CD8 alpha positive alpha beta T cells within the LP and IEL compartments expressed the CD8 alpha homodimer or the CD8 alpha beta heterodimer whereas inductive site CD8 alpha positive alpha beta T cells predominantly expressed the CD8 alpha beta heterodimer.
The majority of gamma delta T cells in the IEL compartment expressed CD8 alpha while gamma delta T cells found in the MLN lack CD8 alpha. While attempting this procedure, it is important to remember to spend enough time and care during tissue isolation steps to limit contamination between intestinal compartments. Following this procedure, isolated lymphocytes can be subjected to ex vivo stimulation, flow cytometry, and other techniques for further phenotypic and functional analysis.
