B.S.S. is supported by NIH grant (R01 AI076457) and funds provided by Stony Brook University. Z.Q. is supported by NIH grant (K12 GM102778).

All animal experiments were conducted in accordance with National Institute of Health guidelines and approved by the Stony Brook University Institutional Animal Care and Use Committee.
NOTE: Ensure that all approvals are granted prior to performing procedures.
1. Solution Preparation
<ol>
	<li>HGPG (HEPES, L-glutamine, penicillin/streptomycin, and gentamycin), 100&#215;
	<ol>
		<li>Mix 59.6 g HEPES (500 mM final), 14.6 g L-glutamine (200 mM final), 1&#215;106 U penicillin (2,000 U/mL final), 1 g streptomycin (2 mg/mL final), and 2.5 mg gentamicin (5 &#181;g/mL final). Add RPMI 1640 to 500 mL. Adjust pH to 7.5 using NaOH (before adjustment, the buffer is yellow/orange with a pH around 6.0). Filter sterilize using a 0.45 &#181;m filter. Aliquot and store at -20 &#176;C for up to 1 year or at 4 &#176;C for up to 1 month.</li>
	</ol>
	</li>
	<li>HEPES-bicarbonate buffer, 10x
	<ol>
		<li>&#8203;Mix 23.8 g HEPES (100 mM final), 21 g NaHCO3 (250 mM final) and add water to 1,000 mL. Adjust pH to 7.2 with HCl. Filter sterilize using a 0.45 &#956;m filter. Store at room temperature. The pH changes over time. Re-adjust pH periodically.</li>
	</ol>
	</li>
	<li>Harvest media (~ 1 bottle/2 mice)
	<ol>
		<li>To 500 mL RPMI 1640, add 25 mL heat-inactivated fetal bovine serum (5% final), and 5 mL 100&#215; HGPG. Store up to 6 months at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Dithioerythritol (DTE) solution (40 mL/small intestine)
	<ol>
		<li>Mix 50 mL 10&#215; Hanks balanced salt solution, Ca2+- and Mg2+-free, 50 mL 10&#215; HEPES-bicarbonate buffer, and 50 mL heat-inactivated fetal bovine serum (10% final). Add 350 mL H2O and 15.4 mg dithioerythritol/100 mL (1 mM final). Prepare this fresh before use.</li>
	</ol>
	</li>
	<li>Ethylenediaminetetraacetic acid (EDTA) solution (50 mL/small intestine)
	<ol>
		<li>Mix 50 mL 10&#215; Hanks balanced salt solution, Ca2+- and Mg2+-free with 5 mL 100&#215; HGPG. Add 445 mL H2O. Add 260 &#181;L 0.5 M EDTA/100 mL (1.3 mM final). Prepare this fresh before use.</li>
	</ol>
	</li>
	<li>Collagenase solution (30 mL/LP)
	<ol>
		<li>To 500 mL RPMI, add 50&#160;mL FBS (10% FBS final), 5 mL 100&#215; HPGP, 1 mL 0.5 M MgCl2 (1 mM final), and 1 mL 0.5 M CaCl2 (1 mM final). Add collagenase, Type I (100 U/mL final). Prepare this fresh before use.</li>
	</ol>
	</li>
	<li>Density gradient (DG) solutions
	<ol>
		<li>Prepare 1&#215; DG stock solution by mixing 90 mL DG stock solution (refer to the Table of Materials) with 10 mL of 10&#215; PBS. Keep sterile at 4 &#176;C.</li>
		<li>Prepare 44% DG solution (8 mL/IEL, 8 mL/LP) by mixing 44 mL of 1&#215; DG stock solution and 56 mL RPMI 1640. Prepare this fresh before use.</li>
		<li>Prepare 67% DG solution (5 mL/IEL, 5 mL/LP) by mixing 67 mL of 1&#215; DG stock solution and 33 mL RPMI 1640. Prepare this fresh before use.</li>
	</ol>
	</li>
</ol>
2. Isolation of the Mesenteric Lymph Nodes, Intestines, and Peyer&#39;s Patches
<ol>
	<li>Euthanize the mouse using carbon dioxide narcosis and secondary cervical dislocation. Place the mouse on its back and spray with 70% ethanol. Use scissors to make a midline incision, and open the skin and abdominal wall to expose the peritoneal cavity.</li>
	<li>Locate the caecum and use forceps to gently pull the caecum down. Use forceps to carefully move the small intestine to the right, exposing the entire MLN chain, which is aligned with the colon. 
	NOTE: A map of the mesenteric lymph nodes chain and lymphatic drainage of the intestines was previously depicted13.</li>
	<li>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 remove the MLN chain from the bottom to the top by tweezing the fat from around the lymph node with another set of forceps.</li>
	<li>Lay the MLN chain on a paper towel moistened with harvest media. Remove mesentery/fat by rolling the chain on the paper tower and pulling the fat off with two sets of forceps. Place the MLN in cold harvest media for later isolation steps. 
	NOTE: (i) Direct handling of the nodes is discouraged. Instead, grasp the mesentery/fat around them. (ii) It is critical to remove as much mesentery/fat as possible to improve cell viability.</li>
	<li>Cut the small intestine below the pyloric sphincter and above the cecum using scissors. 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.</li>
	<li>Place the intestine on a paper towel moistened with harvest media and tease any remaining mesentery/fat off with two sets of forceps. 
	NOTE: (i) Be sure to remove as much mesentery/fat as possible for optimal cell yield and viability. (ii) Keep the intestine moistened throughout this and the following steps by applying harvest media periodically.</li>
	<li>Collect Peyer&#39;s patches by removing them from the intestine with curved scissors. Use a dissecting scope or magnifying glass if needed (beginners); most Peyer&#39;s patches should be visible to the trained eye. Collect 5-11 (typical) Peyer&#39;s patches from the small intestine. Place Peyer&#39;s patches in cold harvest media for later isolation steps. 
	NOTE: Peyer&#39;s patches are small, primarily round bulges in the outer intestinal wall opposite the line of mesentery attachment.</li>
	<li>Use the flat side of forceps and gently slide from duodenum towards ileum to expel fecal content and mucus. Repeat once to remove most of the mucus. 
	NOTE: Incomplete removal of mucus can decrease cell viability and yield. Nevertheless, be sure to slide the intestine gently to avoid stripping villi or damaging the basement membrane.</li>
	<li>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 ~2-cm pieces. Place the intestinal pieces in a 50-mL conical tube with 25 mL cold harvest media for later isolation steps. 
	NOTE: Keeping the scissors moistened with media will help them slide effortlessly through the intestine.</li>
</ol>
3. Isolation of Lymphocytes from the Inductive Sites
<ol>
	<li>Isolate lymphocytes from MLN by gently dissociating through a 70-&#181;m cell strainer using the plunger of a 3-mL syringe. Wash cell strainer with 5 mL of cold harvest media.</li>
	<li>Pellet MLN-cells by centrifuging at 400 &#215; g for 5 min at 4 &#176;C. Resuspend the cell pellet in 1 mL cold harvest media. Count viable cells using trypan blue exclusion. 
	NOTE: Red blood cell lysis buffer can be used to remove red blood cells if bleeding occurred during removal.</li>
	<li>Isolate lymphocytes from Peyer&#39;s patches by incubating Peyer&#39;s patches in a 15-mL conical tube containing 5 mL prewarmed collagenase solution at 37 &#176;C and 220 rpm for 30 min.</li>
	<li>Vortex for 15 s at maximum setting and filter digested tissue and supernatant into a 50-mL conical tube through a 70-&#181;m cell strainer. Gently dissociate the remaining tissue pieces using the plunger of a 3-mL syringe and wash the cell strainer with 5 mL of cold harvest media.</li>
	<li>Pellet Peyer&#39;s patch cells by centrifuging at 400 &#215; g for 5 min at 4 &#176;C and resuspend the cell pellet in 1 mL cold harvest media. Count viable cells using trypan blue exclusion. 
	NOTE: Peyer&#39;s patches isolation may have some contaminating lamina propria lymphocytes and intraepithelial lymphocytes.</li>
</ol>
4. Isolation of Intraepithelial Lymphocytes from the Small Intestine
<ol>
	<li>Wash the pieces of small intestine three times with 25 mL of cold harvest media by inverting the tube 10 times, letting the intestinal pieces settle, and pouring off the supernatant. 
	NOTE: Tissue pieces should readily settle to the bottom of tube. If they do not, it is an indication that too much mesentery/fat remains attached or they are associated with an air bubble.</li>
	<li>Add 20 mL of prewarmed DTE solution to the 50-mL conical tube containing intestine pieces and transfer to a 50-mL siliconized Erlenmeyer flask containing a stir-bar. Stir at 37 &#176;C and 220 rpm on a magnetic stirrer for 20 min. 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-mL conical tube.</li>
	<li>Vortex the tube at maximum setting for 10 s. Transfer the supernatant into a new 50-mL conical tube through a 70-&#181;m cell strainer being careful to keep the tissue pieces in the original tube. Do not discard the supernatant; the supernatant contains intraepithelial lymphocytes. Pellet cells by centrifuging at 400 &#215; g for 5 min at 4 &#176;C. Resuspend the cell pellet in 10 mL cold harvest media and store on ice.</li>
	<li>Add 20 mL of prewarmed DTE solution to the 50-mL conical tube containing intestine pieces and transfer back to the 50-mL siliconized Erlenmeyer flask containing a stir-bar. Stir at 37 &#176;C and 220 rpm on a magnetic stirrer for 20 min. 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-mL conical tube. 
	NOTE: DTE is a reducing agent that enriches intraepithelial lymphocytes.</li>
	<li>Vortex the tube at maximum setting for 10 s. Transfer the supernatant through a 70-&#181;m cell strainer into the previous tube containing cells from the supernatant of the first DTE treatment (from step 4.3). 
	NOTE: The remaining intestinal pieces are used to isolate lymphocytes from the lamina propria. Quality Control: A piece of tissue can be removed and checked histologically to ensure that epithelial cells are present and the basement membrane is intact.</li>
	<li>Pellet cells by centrifuging the supernatant at 400 &#215; g for 5 min at 4 &#176;C. Resuspend the cell pellet in 8 mL of 44% DG solution at room temperature (RT).</li>
	<li>Transfer the 8 mL 44% DG solution/cell suspension into a 17 mm &#248; x 100 mm style 14-mL polypropylene round-bottom tube. Underlay with 5 mL of RT 67% DG solution. Centrifuge at 1600 &#215; g for 20 min at RT without using the brake. 
	NOTE: Tubes may be pretreated with bovine serum to prevent cells from sticking to the tube walls.</li>
	<li>Note that viable cells form a band (buffy coat) at the 44% to 67% interface, dead cells and some epithelial cells are in a mucoid film at the top of the gradient, and the red blood cells are in the pellet. Remove the top layer of the gradient to within ~2 cm of the interface by vacuum aspiration. Use a Pasteur pipette to harvest the buffy coat at the interface and transfer them to a 50-mL conical tube containing 40 mL cold harvest media.</li>
	<li>Pellet cells by centrifuging at 400 &#215; g for 5 min at 4 &#176;C. Resuspend the cell pellet in 1 mL cold harvest media. Count viable cells using trypan blue exclusion.</li>
</ol>
5. Isolation of Lamina Propria Lymphocytes from the Small Intestine
<ol>
	<li>Remove epithelial cells from intestinal tissue pieces by adding 25 mL of prewarmed EDTA solution to the flask containing the remaining intestinal pieces. Stir flask at 37 &#176;C and 220 rpm on a magnetic stirrer for 30 min. Use a large magnet on the outside of the flask to hold the stir-bar and carefully discard the supernatant while keeping intestinal pieces within the flask. 
	NOTE: (i) EDTA is a calcium chelator that targets calcium-dependent junctions and promotes the detachment of intestinal epithelial cells. (ii) The supernatant should be cloudy and may be used to isolate IEC if their collection is desired.</li>
	<li>Repeat step 5.1. 
	NOTE: The supernatant should be less cloudy following this incubation. Quality Control: A piece of tissue can be removed and checked histologically to ensure that intestinal epithelial cells have been removed and the basement membrane is intact. A sample from the supernatant can also be assessed by flow cytometry to confirm that leukocytes (CD45+ cells) are absent.</li>
	<li>Add 50 mL of RT harvest media to the flask. Stir briefly with a magnet. Let intestinal pieces settle and carefully discard the supernatant. 
	NOTE: This step ensures the removal of EDTA prior to collagenase digestion as EDTA inactivates collagenase by chelating the calcium that is critical for collagenase function.</li>
	<li>Add 30 mL of prewarmed collagenase solution to the flask and stir intestinal pieces at 37 &#176;C and 220 rpm on a magnetic stirrer for 45 min.</li>
	<li>Transfer digested tissue and supernatant to a 50-mL conical tube by filtering through a 70-&#181;m cell strainer. Gently dissociate remaining tissue pieces by using the plunger of a 3-mL syringe to mash through the filter. Wash the filter with 10 mL of cold harvest media.</li>
	<li>Pellet cells by centrifuging at 400 &#215; g for 5 min at 4 &#176;C. Resuspend the cell pellet in 8 mL of ambient temperature 44% DG solution.</li>
	<li>Transfer the 8 mL 44% DG solution/cell suspension into a 17 mm &#248; x 100 mm style 14-mL polypropylene round-bottom tube. Underlay with 5 mL of ambient temperature 67% DG solution. Centrifuge the gradients at room temperature and 1600 &#215; g for 20 min with no brake.</li>
	<li>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-mL conical tube containing 40 mL cold harvest media.</li>
	<li>Pellet cells by centrifuging at 400 &#215; g for 5 min at 4 &#176;C. Resuspend the cell pellet in 1 mL cold harvest media. Count viable cells using trypan blue exclusion.</li>
</ol>

<ol>
	<li>Hooper, L. V., Macpherson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+adaptations+that+maintain+homeostasis+with+the+intestinal+microbiota.">Immune adaptations that maintain homeostasis with the intestinal microbiota.</a> Nat Rev Immunol. 10 (3), 159-169 (2010).</li><li>Brandtzaeg, P., Kiyono, H., Pabst, R., Russell, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terminology:+nomenclature+of+mucosa-associated+lymphoid+tissue.">Terminology: nomenclature of mucosa-associated lymphoid tissue.</a> Mucosal Immunol. 1 (1), 31-37 (2008).</li><li>Lefrancois, L., Lycke, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mouse+small+intestinal+intraepithelial+lymphocytes,+Peyer's+patch,+and+lamina+propria+cells.">Isolation of mouse small intestinal intraepithelial lymphocytes, Peyer's patch, and lamina propria cells.</a> Curr Protoc Immunol. , 19 (2001).</li><li>Sheridan, B. S., Lefrancois, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mouse+lymphocytes+from+small+intestine+tissues.">Isolation of mouse lymphocytes from small intestine tissues.</a> Curr Protoc Immunol. , 19 (2012).</li><li>Goodyear, A. W., Kumar, A., Dow, S., Ryan, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+murine+small+intestine+leukocyte+isolation+for+global+immune+phenotype+analysis.">Optimization of murine small intestine leukocyte isolation for global immune phenotype analysis.</a> J Immunol Methods. 405, 97-108 (2014).</li><li>Couter, C. J., Surana, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Flow+Cytometric+Characterization+of+Murine+Small+Intestinal+Lymphocytes.">Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes.</a> J Vis Exp. (111), (2016).</li><li>Koscso, B., Bogunovic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+and+Purification+of+Mouse+Intestinal+Dendritic+Cell+and+Macrophage+Subsets+by+Flow+Cytometry.">Analysis and Purification of Mouse Intestinal Dendritic Cell and Macrophage Subsets by Flow Cytometry.</a> Curr Protoc Immunol. 114, 11-14 (2016).</li><li>Goodman, T., Lefrancois, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+gamma-delta+T-cell+receptor+on+intestinal+CD8++intraepithelial+lymphocytes.">Expression of the gamma-delta T-cell receptor on intestinal CD8+ intraepithelial lymphocytes.</a> Nature. 333 (6176), 855-858 (1988).</li><li>Huleatt, J. W., Lefrancois, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta2+integrins+and+ICAM-1+are+involved+in+establishment+of+the+intestinal+mucosal+T+cell+compartment.">Beta2 integrins and ICAM-1 are involved in establishment of the intestinal mucosal T cell compartment.</a> Immunity. 5 (3), 263-273 (1996).</li><li>Sheridan, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gammadelta+T+cells+exhibit+multifunctional+and+protective+memory+in+intestinal+tissues.">gammadelta T cells exhibit multifunctional and protective memory in intestinal tissues.</a> Immunity. 39 (1), 184-195 (2013).</li><li>Sheridan, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+infection+drives+a+distinct+population+of+intestinal+resident+memory+CD8(+)+T+cells+with+enhanced+protective+function.">Oral infection drives a distinct population of intestinal resident memory CD8(+) T cells with enhanced protective function.</a> Immunity. 40 (5), 747-757 (2014).</li><li>Romagnoli, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+distinct+long-lived+memory+CD4+T+cells+in+intestinal+tissues+after+oral+Listeria+monocytogenes+infection.">Differentiation of distinct long-lived memory CD4 T cells in intestinal tissues after oral Listeria monocytogenes infection.</a> Mucosal Immunol. 10 (2), 520-530 (2017).</li><li>Houston, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lymph+nodes+draining+the+small+intestine+and+colon+are+anatomically+separate+and+immunologically+distinct.">The lymph nodes draining the small intestine and colon are anatomically separate and immunologically distinct.</a> Mucosal Immunol. 9 (2), 468-478 (2016).</li><li>Lorenz, R. G., Newberry, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+lymphoid+follicles+can+function+as+sites+for+induction+of+mucosal+immune+responses.">Isolated lymphoid follicles can function as sites for induction of mucosal immune responses.</a> Ann N Y Acad Sci. 1029, 44-57 (2004).</li><li>Kim, S. K., Reed, D. S., Heath, W. R., Carbone, F., Lefrancois, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+and+migration+of+CD8+T+cells+in+the+intestinal+mucosa.">Activation and migration of CD8 T cells in the intestinal mucosa.</a> J Immunol. 159 (9), 4295-4306 (1997).</li><li>Huleatt, J. W., Lefrancois, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen-driven+induction+of+CD11c+on+intestinal+intraepithelial+lymphocytes+and+CD8++T+cells+in+vivo.">Antigen-driven induction of CD11c on intestinal intraepithelial lymphocytes and CD8+ T cells in vivo.</a> J Immunol. 154 (11), 5684-5693 (1995).</li><li>Van Damme, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+agents+and+enzymes+used+for+the+extraction+of+gut+lymphocytes+influence+flow+cytometric+detection+of+T+cell+surface+markers.">Chemical agents and enzymes used for the extraction of gut lymphocytes influence flow cytometric detection of T cell surface markers.</a> J Immunol Methods. 236 (1-2), 27-35 (2000).</li></ol>

The authors have nothing to disclose.

A detailed protocol is presented for the isolation of lymphocytes from the intestinal mucosal inductive (MLN and PP) and effector (LP and IEL compartment) sites. The protocol has been developed to balance input (time) and output (viability and yield) to maximize productivity and results. The protocol also ensures minimal cross compartmental contamination between LP and IEL compartments.
Several protocols for the isolation of immune cells from the mouse small intestine have been published<sup c...

The gastrointestinal (GI) tract has many folds and protrusions that represents the largest interface separating the internal body and the external environment. The intestinal immune system plays an essential role in maintaining the barrier function of the GI tract. It is constantly exposed to dietary antigens, commensal bacteria, and pathogenic microbes. As such, it must remain tolerant to food antigens and commensal bacteria while preserving the capacity to rapidly generate an effective immune response to enteropathogenic microbes1. The intestinal immune system can be anatomically divided into inductive sites, where na&#239;ve lymphocytes are activated by antigen presenting cells carrying antigens from the intestinal mucosa, and effector sites, where activated lymphocytes exert specific effector functions2. The inductive sites comprise the organized lymphoid structure of Peyer&#39;s patches (PP) that surveys the intestinal lumen directly through the action of specialized M cells and the regional draining mesenteric lymph nodes (MLN). The effector sites consist of the lamina propria (LP), which is the connective tissue below the basement membrane, and the intestinal epithelium, a single cell layer located above the basement membrane that contains intraepithelial lymphocytes (IEL). Lymphocytes are major players of adaptive immunity that mediate protection against infections and cancers and may also contribute to immunopathology in inflammatory diseases. It is important and highly relevant to study lymphocytes in these distinct anatomical mucosal compartments to better understand their induction and effector functions.
A relatively simple and unified protocol for the isolation of lymphocytes from these compartments is needed as the number of investigators exploring immune events occurring in the intestine are accelerating. Several research groups have published protocols that share several similar processes for isolating immune cells from the mouse small intestinal compartments3,4,5,6,7. However, there are several technical differences among them depending on the focus of the individual protocol. For example, with the focus on isolating immune cells from the LP, one protocol examines the impact of various enzymatic digestions on cell viability, cell surface marker expression, and the composition of isolated immune cells5. Another protocol highlights a rapid and reproducible method for isolation of lymphocytes without density centrifugation6. Finally, specific protocols also exist for the purpose of isolating mononuclear phagocytes from different tissue layers of the small intestine7. Here, a highly reproducible protocol that allows sequential isolation of lymphocyte populations from the MLN, PP, LP, and IEL compartment of the small intestine is presented.
We focus on isolating highly purified populations from the LP and IEL compartments, which are largely free of contaminants from other intestinal compartments. This widely used protocol produces a high yield of maximally pure and viable lymphocytes within acceptable time constraints4,8,9,10,11,12. This protocol also ensures the isolation of lymphocytes from the LP and IEL compartment with minimal cross compartmental contamination, allowing a bona fide opportunity to study lymphocytes in these distinct compartments. The isolated lymphocytes can be subjected to further manipulations like flow cytometric analysis or functional analysis. This protocol has been successfully applied to the isolation of lymphocytes from the mouse small intestine and colon during bacterial infections such as Listeria monocytogenes, Salmonella typhimurium, and Yersinia pseudotuberculosis infections and inflammatory conditions such as chemical- and pathogen-induced colitis. This protocol can also be used to isolate innate immune cells such as dendritic cells, macrophages, neutrophils, and monocytes from the mouse small intestine and colon.

<table><tbody><tr><td>HEPES</td><td>Fisher Scientific</td><td>BP310-500</td><td></td></tr><tr><td>L-glutamine&amp;nbsp;</td><td>Sigma-aldrich</td><td>G3126-100G</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Gentamicin</td><td>Life Technologies</td><td>15710-072</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Fisher Scientific</td><td>S318-500</td><td></td></tr><tr><td>RPMI 1640</td><td>Life Technologies</td><td>21870-076</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Fisher Scientific</td><td>S233-500</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Life Technologies</td><td>26140-079</td><td></td></tr><tr><td>10x Hanks&#039; balanced salt solution</td><td>Sigma-aldrich</td><td>H4641-500ML</td><td></td></tr><tr><td>1,4-Dithioerythritol</td><td>Sigma-aldrich</td><td>D9680-5G</td><td></td></tr><tr><td>0.5M EDTA, pH 8.0</td><td>Life Technologies</td><td>15575-020</td><td></td></tr><tr><td>Calsium chloride hexahydrate</td><td>Sigma-aldrich</td><td>21108-500G</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Sigma-aldrich</td><td>M2670-100G</td><td></td></tr><tr><td>Collagenase, Type I</td><td>Life Technologies</td><td>17100-017</td><td></td></tr><tr><td>DG gradient stock solution (Percoll)&amp;nbsp;</td><td>GE Healthcare</td><td>17-0891-01</td><td></td></tr><tr><td>Red Blood Cell Lysis Buffer</td><td>Biolegend</td><td>420301</td><td></td></tr><tr><td>70-&amp;micro;m cell strainer&amp;nbsp;</td><td>Corning</td><td>352350</td><td></td></tr><tr><td>14 mL Polypropylene Round-Bottom Tube</td><td>Corning</td><td>352059</td><td></td></tr><tr><td>Erlenmeyer flask&amp;nbsp;</td><td>Kimble</td><td>26500R-50mL</td><td></td></tr><tr><td>Magnetic stirrer</td><td>Thermo Fisher</td><td>50094596</td><td></td></tr><tr><td>Stir bar</td><td>Fisher Scientific</td><td>14-512-148</td><td></td></tr></tbody></table>

isolating lymphocytes from the mouse small intestinal immune system

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

A schematic representation of the protocol is depicted (Figure 1). Lymphocytes within the intestinal mucosa inductive and effector sites are distinctly organized. Peyer&#39;s patches (PP) and mesenteric lymph nodes (MLN) contain lymphocytes in well-organized T-cell areas and B-cell follicles, whereas the intestinal epithelium contains lymphocytes that are more diffusely distributed. The lamina propria (LP) contains both diffusely distributed lymphocytes and l...

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Isolating Lymphocytes from the Mouse Small Intestinal Immune System

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

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Research

JoVE Journal

Immunology and Infection

52.8K Views.  Stony Brook University. Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer's patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Video: Isolating Lymphocytes from the Mouse Small Intestinal Immune System

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

Biology

Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense against pathogens. It is well established that Peyer&#39;s patches (PPs) have an essential role in the mucosal immune network by hosting several effector T and B cell subsets. A certain fraction of these effector cells, follicular T helper (TFH) and germinal center (GC) B cells are professionalized in the regulation of humoral immunity. Hence, the characterization of these cell subsets within PPs in terms of their differentiation program and functional properties can provide important information about mucosal immunity. To this end, an easily applicable, efficient and reproducible method of lymphocyte isolation from PPs would be valuable to researchers. In this study, we aimed to generate an effective method to isolate lymphocytes from mouse PPs with high cell yield. Our approach revealed that initial tissue processing such as the use of digestive reagents and tissue agitation, as well as cell staining conditions and selection of antibody panels, have great influence on the quality and identity of the isolated lymphocytes and on experimental outcomes.
Here, we describe a protocol enabling researchers to efficiently isolate lymphocyte populations from PPs allowing reproducible flow cytometry-based assessment of T and B cell subsets primarily focusing on&#160;TFH and GC B cell subsets.

In this study, we present a novel and effective protocol for the isolation of lymphocytes from Peyer&#39;s Patches (PPs), which can be subsequently used for in vivo and in vitro functional assays as well as flow cytometric studies of follicular T helper and germinal center B cells.

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense ...

Cancer Research

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response against helminth infections, where it plays an important role in the expulsion of the parasite. In a murine model of Nippostrongylus brasiliensis infection, IL-9 is produced mainly by CD4+ T lymphocytes and innate lymphoid cells found in the lung, small intestine, and draining lymph nodes. Given the technical difficulties involved in the intracellular staining of IL-9, as well as the complexity of isolating hematopoietic cells from the small intestine upon infection, there is a pressing need for a comprehensive but straightforward protocol to analyze the expression of IL-9 in different lymphoid and non-lymphoid tissues in this model. The protocol described here outlines the kinetics of IL-9 produced by CD4+ T cells and innate lymphoid cells in the lung and small intestine, the main organs targeted by N. brasiliensis, as well as in the mediastinal and mesenteric lymph nodes, throughout the infection. In addition, it details the number of larvae needed for infection, depending on the cell type and organ of interest. This protocol aims to assist in the standardization of assays to save time and resources by offering the opportunity to focus on the specific cells, organs, and disease stages of interest in the N. brasiliensis infection model.

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

IL-9-expressing T and ILC2 cells are induced during N. brasiliensis infection, yet their characterization has been largely overlooked in the infected intestine due to their low frequency and differential kinetics. This protocol describes the isolation of these cells from different target organs and confirmation of their identity via flow cytometry at different infection stages.

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

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Przeglądaj więcej filmów

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model