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 border="0" cellpadding="0" cellspacing="0" style="width:712px;" width="713">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">HEPES</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:119px;">BP310-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine&#160;</td>
			<td style="width:123px;">Sigma-aldrich</td>
			<td style="width:119px;">G3126-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Penicillin-Streptomycin</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Gentamicin</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:119px;">15710-072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Sodium Hydroxide</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S318-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">RPMI 1640</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:119px;">21870-076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Sodium bicarbonate</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S233-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Fetal bovine serum</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:119px;">26140-079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">10x Hanks&#39; balanced salt solution</td>
			<td style="width:123px;">Sigma-aldrich</td>
			<td style="width:119px;">H4641-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1,4-Dithioerythritol</td>
			<td style="width:123px;">Sigma-aldrich</td>
			<td style="width:119px;">D9680-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">0.5M EDTA, pH 8.0</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:119px;">15575-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calsium chloride hexahydrate</td>
			<td style="width:123px;">Sigma-aldrich</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride hexahydrate</td>
			<td style="width:123px;">Sigma-aldrich</td>
			<td>M2670-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase, Type I</td>
			<td style="width:123px;">Life Technologies</td>
			<td>17100-017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">DG gradient stock solution (Percoll)&#160;</td>
			<td style="width:123px;">GE Healthcare</td>
			<td style="width:119px;">17-0891-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Red Blood Cell Lysis Buffer</td>
			<td style="width:123px;">Biolegend</td>
			<td style="width:119px;">420301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70-&#181;m cell strainer&#160;</td>
			<td style="width:123px;">Corning</td>
			<td style="width:119px;">352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">14 mL Polypropylene Round-Bottom Tube</td>
			<td style="width:123px;">Corning</td>
			<td style="width:119px;">352059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Erlenmeyer flask&#160;</td>
			<td style="width:123px;">Kimble</td>
			<td style="width:119px;">26500R-50mL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Magnetic stirrer</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:119px;">50094596</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Stir bar</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">14-512-148</td>
			<td></td>
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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...

Watch this Scientific Journal Video about Isolating Lymphocytes from the Mouse Small Intestinal Immune System at JoVE.com

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 ...

isolating-lymphocytes-from-the-mouse-small-intestinal-immune-system

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JoVE Journal

Immunology and Infection

52.3K 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

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

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 ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

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 ...

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has a broad spectrum of applications. Detection of antigens allows for characterization and identification of multiple cell types. Located above the kidneys and encapsulated by a layer of mesenchymal cells, the adrenal gland is an endocrine organ composed by two different tissues with different embryological origins, the mesonephric intermediate mesoderm-derived outer cortex and the neural crest-derived inner medulla. The adrenal cortex secretes steroids (i.e., mineralocorticoids, glucocorticoids, sex hormones), whereas the adrenal medulla produces catecholamines (i.e., adrenaline, noradrenaline). While conducting adrenal research, it is important to be able to distinguish unique cells with different functions. Here we provide a protocol developed in our laboratory that describes a series of sequential steps required for obtaining immunofluorescence staining to characterize the cell types of the adrenal gland. We focus first on the dissection of the mouse adrenal glands, the microscopic removal of periadrenal fat followed by the fixation, processing and paraffin embedding of the tissue. We then describe sectioning of the tissue blocks with a rotary microtome. Lastly, we detail a protocol for immunofluorescent staining of adrenal glands that we have developed to minimize both non-specific antibody binding and autofluorescence in order to achieve an optimal signal.

Here we present a method to isolate adrenal glands from mice, fix the tissues, section them, and perform immunofluorescence staining.

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;