NKS is supported by NIH award K08 AI108690.

All studies were conducted under strict review and guidelines according to the Institutional Animal Care and Use Committee (IACUC) at Harvard Medical School, which meets the veterinary standards set by the American Association for Laboratory Animal Science (AALAS).
1. Horizontal Transmission of Bacteria via Co-housing (Optional)&#160;
<ol>
	<li>To minimize exogenous contamination (particularly if using gnotobiotic mice), practice aseptic technique while assembling sterile disposable cages, using food and water that have both been autoclaved.</li>
	<li>Use ear punches or tags to individually mark 6 week old C57BL/6 mice that harbor different microbiotas and house them in the same cage. Given that mice are coprophagic and eat fecal pellets from the cage floor, microbes will naturally be transmitted horizontally between the mice.</li>
	<li>Co-house mice for &#8805;1 week to allow time for microbial transfer and immunologic change prior to analysis.</li>
</ol>
2. Preparation of a Single Cell Suspension from the Small-intestinal Intraepithelial Layer and Lamina Propria
<ol>
	<li>Preparation of Solutions
	<ol>
		<li>Prepare extraction media (per small intestine): 30 ml RPMI + 93 &#181;l 5% (w/v) dithiothreitol (DTT) + 60 &#181;l 0.5 M EDTA + 500 &#181;l fetal bovine serum (FBS). Add the DTT immediately before use.</li>
		<li>Prepare digestion media (per small intestine): 25 ml RPMI + 12.5 mg dispase + 37.5 mg collagenase II + 300 &#181;l FBS. Add the dispase and collagenase immediately before use.</li>
		<li>For all incubations performed at 37 &#176;C, prewarm solutions to 37 &#176;C.</li>
	</ol>
	</li>
	<li>Euthanize the mouse by CO2 asphyxiation followed by cervical dislocation.</li>
	<li>Place the mouse dorsal side down and spray the abdomen with 70% ethanol. Use scissors to perform a laparotomy by sequentially cutting the skin and then peritoneal fascia along the ventral midline from the pubic symphysis to the xiphoid process, thus exposing the peritoneal cavity.</li>
	<li>Use scissors to separate the small intestine from the stomach by transecting the pyloric sphincter. Gently remove the small intestine from the peritoneum, teasing away the mesenteric fat.
	<ol>
		<li>To fully remove the small intestine, make a second cut at the ileo-cecal junction. Place the isolated small intestine into cold (i.e., 4 &#176;C) RPMI containing 10% FBS to maximize cell viability.</li>
	</ol>
	</li>
	<li>Gently remove large pieces of fat using curved forceps. Use caution to avoid tearing the intestinal tissue itself while trying to remove fat.</li>
	<li>To remove intestinal contents, gently flush intestines with 15 - 20 ml of cold PBS using an 18 G feeding needle affixed to a syringe.</li>
	<li>Use scissors to excise PPs, and place them into cold RPMI containing 5% FBS. PPs are located on the antimesenteric side of the small intestine and appear as a multi-lobulated white mass. Depending on the specific strain, a mouse typically has 8 - 12 PPs.</li>
	<li>Cut the small intestine into 3 - 4 inch segments.</li>
	<li>Remove residual fat by rolling each small-intestinal segment on a paper towel moistened with RPMI, using a dull scalpel to tease the fat away from the tissue.</li>
	<li>Turn the tissue inside out by cannulating the intestinal segments with curved forceps and grasping the distal end of the tissue. Then use a pair of straight forceps to gently remove the tissue segment from the curved forceps (beginning at the proximal end), resulting in the tissue being inverted.</li>
	<li>Place tissue segments in a cup containing 30 ml of extraction media and a stir bar. Secure the lid on the cup, and stir at 500 rpm for 15 min at 37 &#176;C; stirring should be vigorous but not turbulent.</li>
	<li>After incubation, use a steel strainer to separate tissue pieces from the epithelium-containing supernatant, which should appear cloudy. This supernatant contains intraepithelial lymphocytes that can be further analyzed, if desired, by proceeding to filtering the sample in steps 2.18 - 2.23.</li>
	<li>Manually agitate the tissue pieces in RPMI to wash away residual extraction media.</li>
	<li>Place tissue on a dry paper towel and flip it end over end several times to facilitate removal of residual mucus that was not liberated by the extraction medium.</li>
	<li>Place tissue fragments in a 1.5 ml tube with 600 &#181;l of digestion medium.</li>
	<li>Use scissors to mince the tissue until pieces no longer stick to the scissors and the solution appears homogenous. This step is critical to ensure complete enzymatic digestion of the tissue.</li>
	<li>Add the minced small intestine to a cup containing 25 ml of digestion media. Stir at 500 rpm for 30 min at 37 &#176;C. Halfway through the digestion (i.e., at 15 min), pipet up and down with a serological pipet to help break up any large chunks of tissue.</li>
	<li>Filter digested tissue (or epithelial layer from step 2.12 if processing IELs) through a 100 &#181;m cell strainer into a 50 ml tube. Rinse the strainer with 20 ml of RPMI containing 10% FBS.</li>
	<li>Centrifuge the filtered solution at 500 x g for 10 min at 4 &#176;C.</li>
	<li>Carefully decant supernatant and resuspend pellet in 1 ml of RPMI containing 10% FBS.</li>
	<li>Filter resuspended cells through a 40 &#181;m cell strainer into a 50 ml tube. Rinse strainer with 20 ml of RPMI containing 10% FBS.</li>
	<li>Centrifuge filtered solution at 500 x g for 10 min at 4 &#176;C.</li>
	<li>Carefully decant supernatant, and resuspend pellet in 1 ml of RPMI containing 2% FBS.</li>
</ol>
3. Preparation of a Single Cell Suspension from Peyer&#39;s Patches
<ol>
	<li>Transfer excised PPs to a cup with 25 ml of digestion media and a stir bar. Secure lid, and spin at 500 rpm for 10 min at 37 &#176;C.</li>
	<li>Filter digested PPs through a 40 &#181;m cell strainer into a 50 ml tube. If any clumps remain, press them through the strainer using the flat end of the plunger from a 1 ml syringe.</li>
	<li>Rinse strainer with 10 ml of RPMI containing 10% FBS.</li>
	<li>Centrifuge filtered solution at 500 x g for 10 min at 4 &#176;C.</li>
	<li>Carefully decant supernatant, and resuspend pellet in 1 ml of RPMI containing 2% FBS.</li>
</ol>
4. Surface Staining for Flow Cytometry
<ol>
	<li>Aliquot appropriate volume of cells (e.g., 200 &#181;l of LPLs) into a 96-well round-bottom plate.</li>
	<li>Centrifuge at 500 x g for 5 min at 4 &#176;C. Decant supernatant by inverting the plate.</li>
	<li>Resuspend cells in 90 &#181;l of RPMI containing 2% FBS and a 1:100 dilution of anti-CD16/32 (Fc block). Incubate for 10 min at 4 &#176;C.</li>
	<li>Add 10 &#181;l PBS. Centrifuge at 500 x g for 5 min at 4 &#176;C. Decant supernatant by inverting the plate.</li>
	<li>Resuspend cells in 250 &#181;l PBS. Centrifuge at 500 x g for 5 min at 4 &#176;C. Decant supernatant by inverting the plate.</li>
	<li>(Optional) Stain cells with viability dye, if desired, by following the manufacturer&#39;s protocol.</li>
	<li>Resuspend cells in 100 &#181;l 1% formalin to fix the cells. Incubate for 1 hr&#160;in the dark at 4 &#176;C.</li>
	<li>Add 200 &#181;l PBS. Centrifuge at 500 x g for 5 min at 4 &#176;C. Decant supernatant by inverting the plate.</li>
	<li>Resuspend cells in 250 &#181;l PBS. Centrifuge at 500 x g for 5 min at 4 &#176;C. Decant supernatant by inverting the plate.</li>
	<li>Resuspend cells in 200 &#181;l PBS. Analyze cells using a flow cytometer.</li>
</ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+influences+in+inflammatory+bowel+diseases.">Microbial influences in inflammatory bowel diseases.</a> Gastroenterology. 134 (2), 577-594 (2008).</li><li>Tlaskalova-Hogenova, H., 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=Commensal+bacteria+(normal+microflora),+mucosal+immunity+and+chronic+inflammatory+and+autoimmune+diseases.">Commensal bacteria (normal microflora), mucosal immunity and chronic inflammatory and autoimmune diseases.</a> Immunol Lett. 93 (2-3), 97-108 (2004).</li><li>Wen, L., 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=Innate+immunity+and+intestinal+microbiota+in+the+development+of+Type+1+diabetes.">Innate immunity and intestinal microbiota in the development of Type 1 diabetes.</a> Nature. 455 (7216), 1109-1113 (2008).</li><li>Pabst, R., Russell, M. W., Brandtzaeg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+distribution+of+lymphocytes+and+plasma+cells+and+the+role+of+the+gut.">Tissue distribution of lymphocytes and plasma cells and the role of the gut.</a> Trends Immunol. 29 (5), 206-208 (2008).</li><li>Surana, N. K., Kasper, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yin+yang+of+bacterial+polysaccharides:+lessons+learned+from+B.+fragilis+PSA.">The yin yang of bacterial polysaccharides: lessons learned from B. fragilis PSA.</a> Immunol Rev. 245 (1), 13-26 (2012).</li><li>Surana, N. K., Kasper, D. 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No conflicts of interest declared.

We present a protocol for the isolation and flow cytometric characterization of small-intestinal lymphocytes, including the LPLs, IELs, and lymphocytes in the PPs. For those interested in evaluating how changes in the microbiota affect the small-intestinal immune system, we detail the straightforward steps involved in the horizontal transmission of organisms between mice harboring different microbiotas. Although this protocol focuses on the small intestine, the procedure is the same for analysis of the large intestine, w...

The principal task of the small intestine is often considered to be the digestion and absorption of nutrients1. While this metabolic function is clearly essential, the small intestine has an equally significant role in protecting the host from the continual barrage of environmental antigens found within the lumen2. The intestinal tract separates the outside world (e.g., luminal antigens) from the internal environment of the host with an epithelial layer that is only a single cell layer thick. As such, the small-intestinal immune system has the formidable task of balancing its threshold for reactivity, allowing foreign antigens from the diet and commensal microbes to enter the mucosa with minimal, if any, immune response while mounting a robust response against invading pathogens and other &#34;harmful&#34; antigens. Excessive or inappropriate immune responses to these antigens can lead to pathologic disease (e.g., inflammatory bowel disease, type I diabetes, multiple sclerosis) and must be avoided3-6.
Overall, the gastrointestinal tract is thought to represent the largest immune organ in the body, containing over 70% of all antibody-secreting cells7. The small-intestinal immune system is comprised of 3 main compartments&#160;&#8212;&#160;the lamina propria (LP), the intraepithelial layer, and Peyer&#39;s patches (PPs) &#8212;&#160;that each contains a distinctive group of lymphocytes2. The LP lymphocytes (LPLs) are primarily TCR&#945;&#946;+ T cells with ~20% B cells; intraepithelial lymphocytes (IELs) contain very few B cells with more TCR&#947;&#948;+ T cells than TCR&#945;&#946;+ T cells; and PPs, which are secondary lymphoid organs embedded in the small-intestinal wall, contain ~80% B cells. Although each of these anatomical regions has slightly distinct functions and ontological bases, they function in a harmonized fashion to protect the host from pathogenic insults.
Furthermore, there is growing appreciation that the microbiota is a critical determinant for the development of the intestinal immune system, with increasing recognition of the cognate relationship between specific microbes and the ontogeny of particular cell lineages8,9. Moreover, given that education of the intestinal immune system affects immune responses in anatomically distant sites (e.g., arthritis, multiple sclerosis, pneumonia), it has become clear that development of the intestinal immune system is relevant to more disease processes than previously recognized10-12. As such, interest in quantitatively assessing the intestinal immune system has extended beyond host-pathogen interactions to now include host-commensal interactions and the pathogenesis of many systemic diseases as well.
Given the variability of current methods in the isolation of intestinal lymphocytes, a method that is optimized for yield, viability, and consistency while balancing the time required is increasingly critical. Protocols that involve Percoll gradients are time and labor intensive and potentially more prone to human error, leading to variable yield and viability13. Herein, we provide an optimized protocol for the isolation and characterization of lymphocytes from all 3 small-intestinal immune compartments. Additionally, given increasing interest in microbe-induced alterations in the mucosal immune system, we include steps that can be used to allow for the horizontal transmission of microorganisms between mice to assess how these changes quantitatively affect the intestinal immune system.

<table><tbody><tr><td>Sterile Gloves</td><td>Kimberly-Clark</td><td>555092</td><td></td></tr><tr><td>sterile mouse cage</td><td>Innovive</td><td>MS2-AD</td><td>contains lid, cage bottom, and alpha-dri bedding</td></tr><tr><td>Metal feeder</td><td>Innovive</td><td>M-FEED</td><td></td></tr><tr><td>Water bottle</td><td>Innovive</td><td>M-WB-300</td><td></td></tr><tr><td>Card holder</td><td>Innovive</td><td>CRD-HLD-H</td><td></td></tr><tr><td>Autoclavable rodent chow (NIH-31M)</td><td>Zeigler</td><td>4131207530</td><td></td></tr><tr><td>RPMI medium 1640</td><td>Gibco</td><td>11875-119</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Sigma</td><td>D5545-5G</td><td></td></tr><tr><td>0.5 M EDTA (pH 8.0)</td><td>Ambion</td><td>AM9262</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>GemBio</td><td>100-510</td><td></td></tr><tr><td>Dispase II</td><td>Invitrogen</td><td>17105-041</td><td>the concentration in the protocol is based on an activity level of 1.878 U/mg</td></tr><tr><td>Collagenase, type II&amp;nbsp;</td><td>Invitrogen</td><td>17101-015</td><td>the concentration in the protocol is based on an activity level of 245 U/mg</td></tr><tr><td>Dissecting scissors</td><td>Roboz</td><td>RS-5882</td><td></td></tr><tr><td>Feeding needle (18 G, 2&quot; length)</td><td>Roboz</td><td>FN-7905</td><td></td></tr><tr><td>10 ml Syringe</td><td>BD</td><td>305482</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>14190-250</td><td></td></tr><tr><td>Disposable Scalpel (15 blade)</td><td>Miltex</td><td>4-415</td><td></td></tr><tr><td>Curved forceps</td><td>Roboz</td><td>RS-5211</td><td></td></tr><tr><td>Straight forceps</td><td>Roboz</td><td>RS-5132</td><td></td></tr><tr><td>Multi-purpose cups, 120 ml</td><td>VWR</td><td>89009-662</td><td></td></tr><tr><td>Stir bar</td><td>VWR</td><td>58949-062</td><td></td></tr><tr><td>Multi-position stir plate, 9-position</td><td>VWR</td><td>12621-048</td><td></td></tr><tr><td>Stainless steel conical strainer, 3 inch&amp;nbsp;</td><td>RSVP</td><td></td><td></td></tr><tr><td>1.5 ml Tube</td><td>Eppendorf</td><td>0030 125.150</td><td></td></tr><tr><td>100 &amp;mu;m Cell strainer</td><td>Falcon</td><td>08-771-19</td><td></td></tr><tr><td>40 &amp;mu;m Cell strainer</td><td>Falcon</td><td>08-771-1</td><td></td></tr><tr><td>50 ml Conical tube</td><td>Falcon</td><td>352098</td><td></td></tr><tr><td>1 ml Syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>96-well Plate, round-bottom</td><td>Corning</td><td>3799</td><td></td></tr><tr><td>Anti-mouse CD16/32 (Fc block)</td><td>Biolegend</td><td>101320</td><td></td></tr><tr><td>(Optional) Fixable viability dye eFluor 780</td><td>eBiosciences</td><td>65-0865-18</td><td></td></tr><tr><td>10% Formalin, neutral buffered</td><td>Thermo Scientific</td><td>5725</td><td></td></tr></tbody></table>

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.

Flow cytometric analysis of single cell suspensions of small-intestinal lymphocytes should yield a discrete population of cells that have similar forward and side scatter characteristics as splenocytes (Figures 1A and 1B). The lymphocytes may begin to die if the tissue is not maintained at 4 &#176;C during the initial stages of the isolation, resulting in the lymphocyte population having a lower forward scatter and being more difficult to separate from other epithelial an...

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Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

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-flow-cytometric-characterization-murine-small-intestinal

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Immunology and Infection

27.4K Views.  Boston Children’s Hospital. 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. 

Video: Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

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 ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

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.

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 ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow cytometry. The protocol describes how to set up time mating to obtain multiple synchronous dams, the mechanical and enzymatic digestion of the pregnant uterus, the staining of single-cell suspensions, and a FACS strategy to phenotype and discriminate g1 uILCs. Although this method inevitably loses the spatial information of cellular distribution within the tissue, the protocol has been successfully applied to determine uILC heterogeneity, their response to maternal and foetal factors affecting pregnancy, their gene expression profile, and their functions.

This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry.

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow ...

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 ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Biology

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, isolating intestinal lipoproteins is very difficult in vivo because they are first secreted from the small intestine into the mesenteric lymphatics. Chylomicron-containing lymph then empties into the subclavian vein from the thoracic duct to deliver components of the meal to the heart, lungs, and, ultimately, whole-body circulation. Isolating na&#239;ve chylomicrons is impossible from blood since chylomicron triglyceride undergoes hydrolysis immediately upon interaction with lipoprotein lipase and other lipoprotein receptors in circulation. Therefore, the original 2-day lymph fistula procedure, described by Bollman et al. in rats, has historically been used to isolate fresh mesenteric lymph before its entry into the thoracic vein. That protocol has been improved upon and professionalized by the laboratory of Patrick Tso for the last 45 years, allowing for the analysis of these critical lipoproteins and secretions from the gut. The Tso lymph fistula procedure has now been updated and is presented here visually for the first time. This revised procedure is a single-day surgical technique for installing a duodenal feeding tube, cannulating the mesenteric lymph duct, and collecting lymph after a meal in conscious mice. The major benefits of these new techniques include the ability to reproducibly collect lymph from mice (which harnesses the power of genetic mouse models); the reduced anesthesia time for mice during the implantation of the duodenal infusion tube and the lymph cannula; the ability to continuously sample lymph throughout the feeding and post-prandial period; the ability to quantitatively measure hormones and cytokines before their dilution and enzymatic hydrolysis in blood; and the ability to collect large quantities of lymph for isolating intestinal lipoproteins. This technique is a powerful tool for directly and quantitatively measuring dietary nutrient absorption, intestinal lipoprotein synthesis, and chylomicron secretion.

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

The present protocol describes a detailed surgical protocol for isolating flowing intestinal lymph in response to intraduodenal nutrient infusions in mice. This allows for the physiological determination of total intestinal lipid absorption and chylomicron synthesis and secretion in response to various experimental nutrients.

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

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