Isolating Lymphocytes from the Mouse Small Intestinal Immune System

Zhijuan Qiu; Brian S. Sheridan

doi:10.3791/57281

JoVE Journal > Immunology and Infection

Inmunología y contagio

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

Published: February 28, 2018

doi:

10.3791/57281

Zhijuan Qiu¹, Brian S. Sheridan¹

¹Center for Infectious Diseases, Department of Molecular Genetics and Microbiology,Stony Brook University

Summary

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.

Abstract

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'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.

Introduction

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 naïve 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'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.

Protocol

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

HGPG (HEPES, L-glutamine, penicillin/streptomycin, and gentamycin), 100×
1. Mix 59.6 g HEPES (500 mM final), 14.6 g L-glutamine (200 mM final), 1×10⁶ U penicillin (2,000 U/mL final), 1 g streptomycin (2 mg/mL final), and 2.5 mg gentamicin (5 µg/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 µm filter. Aliquot and store at -20 °C for up to 1 year or at 4 °C for up to 1 month.
HEPES-bicarbonate buffer, 10x
1. 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 μm filter. Store at room temperature. The pH changes over time. Re-adjust pH periodically.
Harvest media (~ 1 bottle/2 mice)
1. To 500 mL RPMI 1640, add 25 mL heat-inactivated fetal bovine serum (5% final), and 5 mL 100× HGPG. Store up to 6 months at 4 °C.
Dithioerythritol (DTE) solution (40 mL/small intestine)
1. Mix 50 mL 10× Hanks balanced salt solution, Ca₂⁺– and Mg₂⁺-free, 50 mL 10× 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.
Ethylenediaminetetraacetic acid (EDTA) solution (50 mL/small intestine)
1. Mix 50 mL 10× Hanks balanced salt solution, Ca₂⁺– and Mg₂⁺-free with 5 mL 100× HGPG. Add 445 mL H₂O. Add 260 µL 0.5 M EDTA/100 mL (1.3 mM final). Prepare this fresh before use.
Collagenase solution (30 mL/LP)
1. To 500 mL RPMI, add 50 mL FBS (10% FBS final), 5 mL 100× 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.
Density gradient (DG) solutions
1. Prepare 1× DG stock solution by mixing 90 mL DG stock solution (refer to the Table of Materials) with 10 mL of 10× PBS. Keep sterile at 4 °C.
2. Prepare 44% DG solution (8 mL/IEL, 8 mL/LP) by mixing 44 mL of 1× DG stock solution and 56 mL RPMI 1640. Prepare this fresh before use.
3. Prepare 67% DG solution (5 mL/IEL, 5 mL/LP) by mixing 67 mL of 1× DG stock solution and 33 mL RPMI 1640. Prepare this fresh before use.

2. Isolation of the Mesenteric Lymph Nodes, Intestines, and Peyer's Patches

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.
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.
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.
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.
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.
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.
Collect Peyer's patches by removing them from the intestine with curved scissors. Use a dissecting scope or magnifying glass if needed (beginners); most Peyer's patches should be visible to the trained eye. Collect 5-11 (typical) Peyer's patches from the small intestine. Place Peyer's patches in cold harvest media for later isolation steps.
NOTE: Peyer's patches are small, primarily round bulges in the outer intestinal wall opposite the line of mesentery attachment.
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.
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.

3. Isolation of Lymphocytes from the Inductive Sites

Isolate lymphocytes from MLN by gently dissociating through a 70-µm cell strainer using the plunger of a 3-mL syringe. Wash cell strainer with 5 mL of cold harvest media.
Pellet MLN-cells by centrifuging at 400 × g for 5 min at 4 °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.
Isolate lymphocytes from Peyer's patches by incubating Peyer's patches in a 15-mL conical tube containing 5 mL prewarmed collagenase solution at 37 °C and 220 rpm for 30 min.
Vortex for 15 s at maximum setting and filter digested tissue and supernatant into a 50-mL conical tube through a 70-µm 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.
Pellet Peyer's patch cells by centrifuging at 400 × g for 5 min at 4 °C and resuspend the cell pellet in 1 mL cold harvest media. Count viable cells using trypan blue exclusion.
NOTE: Peyer's patches isolation may have some contaminating lamina propria lymphocytes and intraepithelial lymphocytes.

4. Isolation of Intraepithelial Lymphocytes from the Small Intestine

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.
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 °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.
Vortex the tube at maximum setting for 10 s. Transfer the supernatant into a new 50-mL conical tube through a 70-µm 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 × g for 5 min at 4 °C. Resuspend the cell pellet in 10 mL cold harvest media and store on ice.
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 °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.
Vortex the tube at maximum setting for 10 s. Transfer the supernatant through a 70-µm 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.
Pellet cells by centrifuging the supernatant at 400 × g for 5 min at 4 °C. Resuspend the cell pellet in 8 mL of 44% DG solution at room temperature (RT).
Transfer the 8 mL 44% DG solution/cell suspension into a 17 mm ø x 100 mm style 14-mL polypropylene round-bottom tube. Underlay with 5 mL of RT 67% DG solution. Centrifuge at 1600 × 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.
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.
Pellet cells by centrifuging at 400 × g for 5 min at 4 °C. Resuspend the cell pellet in 1 mL cold harvest media. Count viable cells using trypan blue exclusion.

5. Isolation of Lamina Propria Lymphocytes from the Small Intestine

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 °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.
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.
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.
Add 30 mL of prewarmed collagenase solution to the flask and stir intestinal pieces at 37 °C and 220 rpm on a magnetic stirrer for 45 min.
Transfer digested tissue and supernatant to a 50-mL conical tube by filtering through a 70-µm 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.
Pellet cells by centrifuging at 400 × g for 5 min at 4 °C. Resuspend the cell pellet in 8 mL of ambient temperature 44% DG solution.
Transfer the 8 mL 44% DG solution/cell suspension into a 17 mm ø 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 × g for 20 min with no brake.
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.
Pellet cells by centrifuging at 400 × g for 5 min at 4 °C. Resuspend the cell pellet in 1 mL cold harvest media. Count viable cells using trypan blue exclusion.

Representative Results

A schematic representation of the protocol is depicted (Figure 1). Lymphocytes within the intestinal mucosa inductive and effector sites are distinctly organized. Peyer'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 lymphocytes contained within organized lymphoid structures like isolated lymphoid follicles (ILF) and cryptopatches. Each intestinal immune compartment is also typified by a unique composition of lymphocyte subsets (Figure 2). The MLN is predominately composed of αβ T cells (~70%), while PP lymphocytes are mostly B cells (~80%). LP lymphocyte composition is largely strain-dependent but primarily composed of αβ T cells and B cells. Distinct from the other intestinal compartments, lymphocytes residing in the epithelium are mainly γδ T cells (~60%) and αβ T cells with essentially no B cells. Distinct proportions of αβ T cell subsets also exist within distinct anatomic locations of the intestines. The αβ T cells in the LP are mainly CD4⁺ T cells (~70%), while CD8α⁺ T cells (~85%) predominate in the intraepithelial lymphocytes (IEL) compartment. CD8α⁺ αβ T cells within the LP and IEL compartments express the CD8αα homodimer or the CD8αβ heterodimer whereas inductive site CD8α⁺ αβ T cells predominately express the CD8αβ heterodimer (Figure 3). The majority of γδ T cells in the IEL compartment also express CD8α, while γδ T cells found in the MLN lack CD8α (Figure 3). Regardless of generalities, the composition of lymphocyte subsets can vary between mouse strains and may become altered with age or disease. As such, a careful analysis of lymphocyte populations within the intestinal mucosa at steady-state should be performed in the background strain utilized for the experimental manipulations.

Cell yield can vary greatly depending on the strain and age of the mice, and experimental conditions used. Single, live cells can be counted on a hemocytometer or an automated cell counting machine using trypan blue exclusion. The number of lymphocytes are calculated as: Cell count × CD45⁺ cells (%) in Gate 3 × Lymphocytes (%) in Gate 4 (Figure 2). Approximately 15 × 10⁶ MLN lymphocytes, 5 – 10 × 10⁶ PP lymphocytes, 2 – 5 × 10⁶ LP lymphocytes, and 3 – 6 × 10⁶ IEL can be obtained from an 8- to 12-week-old naïve C57Bl/6 or Balb/c mouse. Alternatively, flow-based counting beads can be used to quantify cell populations with the caveat that cell loss occurs during the staining process. Ultimately, consistency in counting technique must be maintained within the lab to ensure that appropriate comparisons can be made across experiments.

In this protocol, DTE treatment is used to enrich IEL, which is followed by EDTA treatment to remove IEC for optimal isolation of highly enriched lymphocytes. Two DTE treatments are sufficient to isolate >95% of IEL. A very small number of lymphocytes can also be found in the EDTA treatment. These lymphocytes resemble a mixture of IEL and LP lymphocytes (Figure 4). A comparison of DTE treatment and combined DTE-EDTA treatment to isolate IEL has also been performed. Combined DTE-EDTA treatment increases IEC contamination in the IEL preparation (Figure 5) and reduces final yields of live immune cells after density gradient centrifugation. Density gradient centrifugation is recommended to isolate lymphocytes from the intestinal compartments. It removes dead cells, many epithelial cells and mucus, promoting the isolation of highly enriched lymphocytes and drastically decreasing flow cytometry acquisition times (Figure 6).

Figure 1. Flow chart of isolation protocol. DTE, dithioerythritol. EDTA, ethylenediaminetetraacetic acid. Please click here to view a larger version of this figure.

Figure 2. A flow cytometry gating strategy for lymphocyte populations from intestinal tissues. Single cell suspensions prepared from the mesenteric lymph nodes (MLN), lamina propria (LP), Peyer's patches (PP) and intraepithelial lymphocytes (IEL) compartment were stained with fixable viability dye (e.g. Live/Dead) and the following fluorescent-labeled antibodies: CD45 for identification of hematopoietic cells, CD19 for B cells, CD3ε for T cells, T-cell receptor (TCR)β for αβ T cells, TCRδ for γδ T cells, CD8α for CD8α⁺ αβ T cells and CD4 for CD4⁺ αβ T cells. Gate 0 showed forward scatter (FSC)-A/side scatter (SSC)-A properties. Gate 1 identified single cells. Gate 2 identified live cells. Gate 3 identified hematopoietic cells. Gate 4 identified lymphocytes based on FSC-A/SSC-A properties. Gate 5 identified T cells and B cells. Gate 6 identified αβ T cells and γδ T cells among total T cells. Gate 7 identified CD8α⁺ and CD4⁺ T cells in the αβ T cell population. Percentages (in red font) within pseudocolor plots correspond to the frequency of cells within the indicated gate among the preceding parental population. Gate 0 and gate 1 display total events. Please click here to view a larger version of this figure.

Figure 3. The phenotype ofCD8α⁺ αβ T cells and γδ T cells in intestinal tissues. The expression of CD8β by CD8α⁺ αβ T cells and the expression of CD8α by γδ T cells are depicted. Percentages (in red font) within histograms correspond to the frequency of cells that express the indicated marker amongst the indicated parental population. Please click here to view a larger version of this figure.

Figure 4. The composition of lymphocytes from DTE, EDTA, and collagenase treatments. The parental gating strategy is the same as in Figure 2. Gate 5 identified T cells and B cells. Gate 6 identified αβ T cells and γδ T cells among total T cells. Gate 7 identified CD8α⁺ and CD4⁺ T cells in the αβ T cell population. Percentages (in red font) within pseudocolor plots correspond to the frequency of cells within the indicated gate among the preceding parental population. Lymphocytes from EDTA isolation bear resemblance to a mixture of IEL and LP lymphocytes isolated from DTE and collagenase treatments, respectively. Please click here to view a larger version of this figure.

Figure 5. A comparison of DTE treatment and combined DTE-EDTA treatment for the isolation of IEL. DTE treatment was performed according to this protocol. IEL were collected after DTE treatments and prior to EDTA treatments. Combined DTE-EDTA treatment was performed at the same concentrations used in this protocol but at the same time. The FSC-A/SSC-A properties, viability and the percentage of CD45⁺ hematopoietic cells are shown. Please click here to view a larger version of this figure.

Figure 6. The impact of density gradient centrifugation on the purity of IEL and LP lymphocyte preparations. Single cell suspensions isolated according to this protocol were subjected to density gradient centrifugation or analyzed by flow cytometry directly. The FSC-A/SSC-A properties, viability and the percentage of CD45⁺ hematopoietic cells are shown. Please click here to view a larger version of this figure.

Discussion

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 published³^,⁴^,⁵^,⁶^,⁷. Most of these protocols focus on isolating immune cells from the LP. The intestinal epithelium is a distinct compartment from the LP that is directly exposed to the external environment and consists of a unique composition of lymphocytes. Recent approaches have sought a more global analysis of immune function in the intestines by examining distinct intestinal compartments simultaneously to understand the unique aspects of immune responses occurring in these compartments and the relationships between them. Therefore, a reliable protocol for the isolation of immune cells from all these compartments is desired. Two DTE treatments are sufficient to isolate >95% of IEL and some IEC. EDTA targets calcium-dependent junctions and is efficient to detach IEC. Therefore, the supernatant from the subsequent EDTA treatments comprise mostly IEC. A very small number of lymphocytes can be found in the supernatant after EDTA treatment; however, about 20-fold more lymphocytes are obtained from the DTE fraction. Moreover, lymphocytes from the EDTA treatment resemble mixed populations of IEL and LP lymphocytes (Figure 4), which suggests LP contamination from villi and crypt damage. Therefore, in this protocol, IEL are collected from the DTE treatment prior to EDTA treatment, maximizing purity by minimizing cross-compartment contamination. Other protocols use sequential dithiothreitol (DTT)-EDTA treatments or combined DTT-EDTA treatment for the isolation of IEL⁵^,⁶^,⁷. DTT is an epimer of DTE and both act as reducing agents. In these protocols, DTT is reported to only remove mucus and EDTA is used to isolate IEL. Although the effect of DTT and DTE was not directly compared, IEL after DTE treatment or combined DTE-EDTA treatment was evaluated. Not surprisingly, combined DTE-EDTA treatment increases IEC contamination in the IEL preparation (Figure 5) and the presence of large numbers of IEC decreased IEL yields after density gradient centrifugation. As EDTA can damage some villi and crypts, combined DTE-EDTA treatment may also result in contamination of LP lymphocytes in the IEL preparation or loss of LP cells from the LP isolation. Moreover, as >95% of IEL were isolated from the DTE fraction, separate DTE and EDTA treatments are preferred. Some protocols isolate lymphocytes without using density gradient centrifugation to enrich immune cells⁶. Density gradient centrifugation takes additional time and loss of some cells may occur. However, it removes dead cells, epithelial cells and mucus, leading to highly enriched lymphocyte populations (Figure 6), which helps to enhance the detection of small populations of cells and substantially decreases acquisition time for flow cytometry. Density gradient centrifugation has reported to result in altered ratio of mononuclear phagocyte subsets⁷; however, lymphocyte subset composition appears normal. It is particularly helpful to perform density gradient centrifugation for the isolation of IEL. Overall, a complete protocol is presented to isolate highly enriched and optimally viable lymphocytes from all the intestinal tissues including the LP and IEL with minimal cross-compartmental contamination.

Substantial time and care must be taken during tissue isolation steps to limit contamination between preparations. Incomplete removal of PP can greatly skew LP lymphocytes. ILF can also contaminate LP preparations. However, ILF cannot be seen by the naked eye and therefore cannot be surgically removed prior to digestion. Different mouse strains have different numbers of ILF¹⁴, which may greatly contribute to the variable composition of LP lymphocytes between mouse strains. When removing mucus, be sure to gently slide the intestine to avoid damage to the basement membrane and LP, which could result in LP contamination of IEL. One indication of contamination is the presence of large numbers of B cells in the IEL preparation. IEL contain few B cells (~1% or less), whereas LPL can have up to 60% B cells depending on the mouse strain. CD19 but not B220 should be used to identify B cells in the LP and IEL compartment, as intestinal T cells may also express B220¹⁵. Furthermore, many intestinal T cells may express traditional macrophage and DC markers such as CD11b and CD11c, respectively¹⁶. Therefore, proper dump gating strategies should be utilized to ensure appropriate analysis.

Steps may be modified to accommodate researchers' needs. Mechanical digestion may be sufficient for PP lymphocyte isolation at steady state or collagenase digestion of lymph nodes may facilitate isolation of some subsets of leukocytes during inflammation. The type of collagenase used may have substantial impact on cell surface marker expression and the viability and purity of LP lymphocytes⁵^,¹⁷. The intestine may be cut into small pieces for collagenase digestion, which may increase cell yield. However, it also reduces the viability. The incubation time with collagenase may be optimized to maximize the yield while minimizing the cell death, leading to maximum recovery of live cells. Different labs use slightly different density gradients to isolate lymphocytes. A well-established 44%/67% density gradient protocol is presented here. Researchers may test different density gradients to best suit their individual needs. If a larger number of lymphocytes are desired, two small intestines may be combined for DTE treatments, EDTA treatments and collagenase digestion. If more than two intestines are needed for examining extremely rare events, then it is recommended that single cell suspensions are pooled after isolation.

Isolated lymphocytes can be used for further phenotypic, functional, and genomic analysis. When performing flow cytometric analysis, it is highly advisable to always use a viability dyes and anti-CD45 antibody to exclude dead cells and non-hematopoietic cells, which will greatly enhance the analysis. If pure lymphocyte populations are needed, cell sorting of CD45⁺ cells along with other markers of choice can be performed. Panning on anti-CD8α-coated plates can also be used to isolate pure T cells from IEL preparation as majority of these T cells express CD8α. Panning or cell sorting is generally required for culture of IEL as contaminating epithelial cells can inhibit IEL growth. Feeder cells such as naïve splenocytes can also be added to promote IEL survival in culture. If feeders are used, it is important to utilize congenically mismatched cells to distinguish populations. Bacterial contamination is rarely a problem in these short term functional studies as harvest media contains penicillin, streptomycin and gentamicin. In addition, numerous washing steps and density gradient centrifugation remove most bacteria.

The isolation process is time consuming and demands a balance of care, precision and speed for optimal results. Enough time must be spent to ensure proper manipulation of each step while minimizing the time between mouse sacrifice and obtaining a single cell suspension to maximize cell viability and yield. This is a delicate balance that is also a bottleneck for beginners trying to obtain consistency in isolating lymphocytes with high viability and yield. An entire isolation process starting from mouse euthanasia to obtaining a counted single cell suspension takes an experienced researcher approximately 8 h for 8 mice. It is advised to obtain assistance for more than 8 mice to generate high quality and reproducible results. It is important to use care when comparing lymphocytes from these tissues with lymphocytes from the spleen, blood, or other lymphoid tissues as the extensive incubations may lead to artificial changes associated with the isolation procedure¹⁷. Routine comparisons of LP and IEL populations to similarly treated lymphoid tissues would assess whether the marker of interest is modulated during lymphocyte isolation from intestinal tissues. Similar issues are also a major concern when comparing RNA profiles from intestines with those from lymphoid tissues.

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

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).

Materials

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

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Qiu, Z., Sheridan, B. S. Isolating Lymphocytes from the Mouse Small Intestinal Immune System. J. Vis. Exp. (132), e57281, doi:10.3791/57281 (2018).