Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue

Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue Benno Weigmann, Ingrid Tubbe, Daniel Seidel, Alex Nicolaev, Christoph Becker & Markus F Neurath I. Med. Clinic, Immunological Laboratory, Johannes-Gutenberg University, Obere Zahlbacherstrasse 63, 55131 Mainz, Germany. Correspondence should be addressed to B.W. (bweigm@uni-mainz.de). Published online 20 September 2007; doi:.38/nprot.2007.315 PROTOCOL Studies on colonic cells in the lamina propria (LP) of mice are important for understanding the cellular and immune responses in the gut, especially in inflammatory bowel diseases (such as morbus crohn and colitis ulcerosa). This protocol details a method to isolate LP cells and characterize freshly isolated cells by quality control experiments to obtain cells that can be used for further investigations. After different steps of digestion of the tissue using collagenase, DNase and dispase, the resulting cells are purified using Percoll gradient. The success of the isolation can be analyzed by cell viability test (Trypan Blue exclusion test) and by flow cytometric analysis to assess apoptosis. Finally, the isolated cells can be used for further investigations like comparative studies of mrna expression, cell-proliferation assay or protein analysis. This protocol can be completed within 6 7 h. INTRODUCTION The intestinal mucosal immune system is composed of three major lymphoid areas: the LP, which lies just underneath the basement membrane in the intestinal villi; the intraepithelial compartment, which contains the intraepithelial lymphocytes (IELs) and is located just above the basement membrane, between the columnar epithelial cells; and and lymphoid nodules (akin to lymph nodes) embedded in the gut wall and Peyer s patches (PPs), separated from the LP and IELs. The LP, PP and IEL lymphoid populations form a complex, interconnected network that responds to immunological diseases in the intestine. LP T cells mostly express ab T-cell receptor (TCR) and possess a memory/activated phenotype characterized by the following surface markers: CD45RO/45RB lo, CD44 hi, L-Sel lo and ab hi (ref. 1). LP T cells respond poorly in vitro to antigen or TCR stimuli but do secrete large amounts of interferon-g, interleukin-4 and interleukin-5 upon stimulation 2. The IELs of the intestine are believed to originate from thymus-dependent and extrathymic or thymus-independent precursors in the gut wall 3. The thymus-dependent population consists of T cells that bear CD4 or heterodimeric CD8ab molecules, Thy-1 and ab gd TCR, whereas the thymus-independent population expresses either ab or gd TCR, and the majority of these cells bear CD8 homodimeric aa molecules, a few are double negative and only some express Thy-1, however. Local cells in colonic tissue can dramatically induce or influence immunological responses. To analyze the role of these cells in different disease states, viable murine intestinal mucosal lymphoid mononuclear cells need to be isolated. Protocols to isolate intestinal cells were initially established by several researchers 4,5. More recently, improved enzyme purifications have allowed better and more distinct isolation of cell populations 6 11. Alternatively, the Medimachine (Dako, BD) is an automated sample preparation system for the mechanical disaggregation of solid tissue. The advantage of this system is that it can be used without enzymatic digestion, but cell numbers can be insufficient for all further experiments. The Medimachine was mainly developed for isolation of cells for flow cytometric analysis, but is used for isolation of lamina propria mononuclear cells (LPMCs) from the colon in some laboratories. Figure 1 shows a simplified flow diagram of the protocol. The cells are isolated as follows: the intestine is removed from mesenteric fat tissue, PPs are excised and the colon is opened longitudinally and cut into pieces. After incubation with EDTA and dithiothreitol (DTT) in Hanks balanced salt solution (HBSS), vortexing and passing through a cell strainer, the suspension of epithelial cells, villus cells, subepithelial cells and IELs is removed. The remaining LP with muscle layer is collected. After several incubations with digestion solution containing collagenases, DNases and dispases, the suspension is subjected to Percollgradient separation. Collagenases from Clostridium histolyticum are used for the disaggregation of tissue and for the preparation of single-cell suspensions. This enzyme breaks down the native collagen that holds the tissue together. Often, collagenases are a Colon Mucosa LP, muscle layer Digestion with collagenase, DNase, dispase Lymphoid cell suspension LPMCs Mechanical dissection of intestinal layers Purification with Percoll gradient Purified concentrated cell suspension Shaking in HBSS, EDTA, DTT at 37 C for 20 min Epithelial cells, villus cells, subepithelial cells, IELs Crypt epithelium IELs Sediment for 15 min at 37 C Figure 1 Schematic of the procedure for isolation of colonic lymphoid cells. NATURE PROTOCOLS VOL.2 NO. 2007 2307

mixture of several different enzymes. The best choice is the one with high collagenase activity and very low tryptic activity because of subsequent analysis of the resulting cells in the procedure. Some companies offer different products with a balanced mixture of collagenases. The dispase that we recommend you to use, also known as neutral protease, is an amino-endopeptidase from Bacillus polymyxa. The advantage of this dispase is that it does not damage the cell membrane and prevents unwanted cell clumping. DNase is included as this also reduces cell clumping. After the cells are collected, they can be stained with antibodies using flow cytometry. If necessary, cells can be incubated with beads conjugated to antibodies for the further concentration of individual cell populations. For example, purification of dendritic cells from the colon can be done with CD11c beads using immunomagnetic sorting or with fluorescein isothiocyanate or fluorescence stainingconjugated CD11c Abs by flow sorting. The resulting cells can be used for comparative stimulation assays, proliferation assays, isolation of mrna, PCR experiments or protein isolation. MATERIALS REAGENTS. Mice! CAUTION Experiments involving rodents must conform to national and institutional regulations.. 1 Phosphate-buffered saline (PBS). 1 HBSS. Fetal calf serum (FCS). 0 U ml 1 penicillin, 0 U ml 1 streptomycin, 5 mg ml 1 amphotericin B and mg ml 1 gentamycin m CRITICAL These antibiotics should be added to all media to avoid bacterial contamination. EQUIPMENT. Scissors. Forceps. Petri dishes. 40 and 0 mm cell strainers (Falcon; BD). 15 and 50 ml tubes. Thermal incubator with rotation unit. Cell culture centrifuge (e.g., Megafuge 1.0R; Heraeus). Pasteur pipette. Pipetteboy. ph-meter. Cell culture equipment (e.g., lamina flow bench) PROCEDURE 1 Remove the intestines of dead mice and cut into 4 5 cm pieces. Place in ice-cold 1 PBS. m CRITICAL STEP This work should be done in a sterile flow hood. 2 Clear the intestine of feces by holding the intestine with forceps and flushing with a syringe filled with 1 PBS. Resect residual mesenteric fat tissue and PPs (Fig. 2a). 3 Open the intestine pieces longitudinally, cut into 1 cm pieces and wash in ice-cold 1 PBS (Fig. 2b). 4 Incubate the pieces of intestine in 5 ml of predigestion solution for 20 min at 37 1C under slow rotation (40g) in a thermal incubator in a 50 ml tube. REAGENT SETUP Predigestion solution 1 HBSS containing 5 mm EDTA and 1 mm DTT. Digestion solution Dissolve 0.05 g of collagenase D (Roche), 0.05 g of DNase I (Sigma) and 0.3 g of dispase II (Roche) in 0 ml of 1 PBS.! CAUTION Use gloves while preparing and using because the enzymes and DTT can affect the skin. m CRITICAL Prepare a fresh solution just before use and incubate for a few minutes at 37 1C. Fluorescence-activated cell sorting (FACS) buffer Mix 3% (vol/vol) FCS in 1 PBS. Percoll 40% (vol/vol) Use 42.01 ml of Percoll separation solution with 1.124 g ml 1 density and dilute with 57.99 ml of 1 PBS. Percoll 80% (vol/vol) Use 79.83 ml of Percoll separation solution with 1.124 g ml 1 density and dilute with 20.17 ml of 1 PBS. a 5 Pass the remaining pieces through a 0 mm cell strainer or 0 mm nylon mesh; use forceps to dissect residual internal layers from the tissue. Put the pieces together into fresh predigestion solution. The flow-through contains colonic content with epithelial cells and can be decanted or stored for the isolation of IELs. b 6 Again incubate the pieces for 20 min at 37 1C under slow rotation in a 50 ml tube. By shaking the tube during incubation, epithelial cells are disrupted from the mucosa. 7 Pass the pieces through a 0 mm cell strainer. Using scissors, cut the remaining tissue into 1 mm pieces. Wash off the remaining EDTA with 1 PBS. Collect the tissue into 50 ml tubes containing 5 ml of digestion solution. Digest by incubating the pieces at 37 1C for 20 min under slow rotation. 8 After incubation, vortex the cell solution intensely for 20 s and pass through a 40 mm cellstrainersetovera50mltube. Figure 2 Demonstration of the procedure to handle the colon during preparation. (a) Demonstration of how to flush the intestine with a syringe to remove feces. (b) Demonstration of how the colon is opened longitudinally and cut into small pieces. 2308 VOL.2 NO. 2007 NATURE PROTOCOLS

BOX 1 TRYPAN BLUE EXCLUSION TEST Trypan Blue is a vital dye. The reactivity of Trypan Blue is based on the fact that it does not interact with the cells unless the membrane is damaged. All cells that exclude Trypan Blue are viable. Reagents: 1 PBS, Trypan Blue solution (0.4% (wt/vol) in HBSS).! CAUTION Use gloves while preparing and using because Trypan Blue is carcinogenic. Materials: Microscope, hemocytometer. Procedure: 1. Resuspend the cells in an appropriate volume of 1 PBS (e.g., ml). 2. Place 15 ml on the hemocytometer (between the counting slide and the coverslip). 3. Count the cells under the microscope. The grid on the hemocytometer can be seen under magnification. Count the cells in all four outer quadrants of the grid. Divide this number by 4 to determine the average number of cells in one quadrant. 4. The number of total cells is calculated by multiplying the average number of cells per quadrant by the dilution factor (e.g., 1:). Then multiply this number by,000 to calculate the number of cells in 1 ml of cell suspension. After this, multiply this number by the volume (ml) of the cell suspension. 5. Combine 90 ml of 0.4% Trypan Blue solution with ml of the cell suspension. 6. Mix thoroughly and incubate for 3 min at 20 1C. 7. Count the number of unstained cells on the hemocytometer under the microscope as described in Step 3. 8. Calculate the number of unstained cells as described in Step 4. 9. Determine the percentage of viable cells by dividing the number of unstained cells by the total number of cells and multiplying by 0. 15 Carefully collect the cells in a new 15 ml tube using a Pasteur pipette and add 1 PBS. 16 Centrifuge the cell suspension for min at 500g at 20 1C. 17 Resuspend the cells immediately in FACS buffer. 18 To test the viability and the number of extracted cells, perform a quick Trypan Blue exclusion test (Box 1). More time-intensive, but more precise, is a flow cytometric analysis to discriminate between apoptotic and dead cells (Box 2). 19 If desired, LP T cells can be further isolated by using CD4-antibody-coated paramagnetic beads as described by the manufacturer (Miltenyi-Biotech). 20 Viable LP cells can be used in cell culture to obtain cytokine-containing supernatant or cytospins or flow cytometric analysis of surface antigens. Furthermore, the cells can be used for proliferation assays, isolation of mrna to perform PCRs or protein isolation for western blot experiments. 9 Collect all the pieces and place them into 50 ml tubes with fresh digestion solution. Steps 8 and 9 should be repeated as long as connective tissue is visible in the strainer, normally 2 to 3 times. Ideally, all gut pieces should be digested to invisible small pieces. Combine the supernatants from all digestion steps in a fresh 50 ml tube and centrifuge for min at 500g at 20 1C (1,200 r.p.m. using the Megafuge 1.0R). Discard the supernatant. m CRITICAL STEP For isolation of IELs, the flow-through from Step 5 is allowed to sediment for 15 min at 20 1C. The pellet contains mainly crypts and can be used for further purification similar to that of LPMCs with Percoll gradient. 11 Resuspend the pellet in cold FACS buffer and centrifuge the cells at 500g for min at 20 1C. 12 Discard the supernatant and resuspend the pellet in ml of the 40% fraction of the Percoll solution. 13 Using a Pasteur pipette, carefully overlay the cell suspension on top of 5 ml of the 80% fraction of the Percoll solution in a 15 ml tube. 14 Centrifuge the 40/80 Percoll gradient for 20 min at 1,000g (2,400 r.p.m. in the Megafuge 1.0R) at 20 1C withoutbrakes. Following centrifugation, the LPMCs should be visible in a white ring at the interphase of the two different Percoll solutions. Contaminated erythrocytes should be visible in a red ring below the interphase. At the top is a layer of some epithelial cells and at the bottom lies a pellet of debris and dead cells. BOX 2 PROPIDIUM IODIDE/ANNEXIN V ANALYSIS This method is a combination of PI staining 12 and Annexin V staining. The loss of plasma membrane is one of the features of apoptotic cells. The membrane phospholipid phosphatidylserine is then translocated from the inner to the outer plasma membrane. Annexin V is a phospholipid-binding protein that binds to cells with exposed phosphatidylserines and can be conjugated with FITC dye. FITC can be detected on an FL1 axis, whereas PI is measured on FL3. Annexin V single-positive cells are apoptotic cells. PI/Annexin V double-positive cells are necrotic/dead cells. Materials: 2 mg ml 1 PI (BD) in 1 PBS, Annexin V (20 mg ml 1 ) conjugated with FITC (BD), HEPES/Ca 2+ buffer, polystyrene culture tubes, flow cytometer. Procedure: 1. Resuspend 0.5 6 cells in 0.5 ml of HEPES/Ca 2+ buffer. 2. Add 1 ml of PI solution to cells and 1 ml Annexin V FITC. 3. Vortex the cells and incubate for min in the dark on ice. 4. Add 400 ml of HEPES/Ca 2+ buffer. 5. Analyze cells on the flow cytometer with excitation at 488 nm. NATURE PROTOCOLS VOL.2 NO. 2007 2309

TIMING The duration of the procedure depends on the amount of intestine and the ability of the preparator. Normally, the whole procedure, comprising two groups with four colons each, can be completed in less than 6 h without analysis. Specifically, the critical steps, that is, the preparation of the tissue and the overlaying of the Percoll gradient, require a lot of time. Step 9 If the cell suspension clumps together, increase the amount of DNase I. After lysing cells, long DNA molecules remain in cell suspension and stick cells together. To prevent this, DNase I is added. If the cell suspension clumps together, increase the amount of DNase I. The efficacy of the collagenases can vary greatly between manufacturers, and therefore it is normal to test the batch before performing large-scale LPMC preparations. Some batches of collagenases can vary significantly in specific activity, too. Moreover, before purchasing larger quantities, new brands of collagenases should be tested to ensure that they are not toxic to the cells. Different batches of DNase should be tested to determine the optimum quantity to be used for efficient digestion of tissue. Step 16 If, after Percoll gradient centrifugation, only a small ring or no ring is visible but a big cell pellet is present at the bottom of the tube, try to extract the pellet and resuspend cells immediately in a new 40% Percoll solution. Step 17 If the output of the cells before the preparation of the Percoll gradient is too low, try to cut the pieces into even smaller pieces. This helps to increase the surface area exposed to the digestion solution, enabling the cells to be set free and reducing the amount of aggregated tissue left on the filter at the end of the digestion step. a Positive cells (%) b Positive cells (%) 60 50 40 30 20 0 60 50 40 30 20 0 CD4 + CD8 + CD11b + CD11c + CD4 + CD8 + CD11b + CD11c + Figure 3 Isolation of LPMCs from B/6 mice as described in PROCEDURE. Cells were incubated with antibodies against CD4, CD8, CD11c and CD11b to characterize the subpopulations and then measured by flow cytometry. There were 44% CD11c + cells and 12% CD11b + cells. Lower amounts with 7% represent CD4 + and approximately 25% CD8 + T cells. (a) Summary of the number of positive cells from four experiments. (b) Results from LPMCs isolated from the inflamed colon of B/6 mice (transfer colitis experiment) and analyzed by flow cytometry. In b, greater number of CD4 + T (40%) and less number of CD8 + T cells (7%) can be observed. Step 18 Sometimes, after FACS analysis of the resulting LPMCs, the ratio of apoptotic/necrotic cells is too high. This can be due to the fact that the procedure takes quite a long time. Collagenase activity can cause cell death at high concentrations. If this happens, use less collagenase or more FCS to stabilize the cells for further treatment. Buffers should be prewarmed to the optimal temperature of the enzymes. Furthermore, the use of EDTA for a longer time in the medium during isolation is able to reduce the viability of LPMCs. CD4 CD11c Figure 4 Immunofluorescence of cryosections of colon from B/6 mice performed using the Tyramide signal amplification Cy3 system (Perkin-Elmer) and a fluorescence microscope (Olympus). In brief, cryosections were fixed in 4% paraformaldehyde for 20 min at 20 1C in a humid chamber, followed by sequential incubation with avidin/biotin (Vector Laboratories) and proteinblocking reagent (Dako Corp.) to eliminate unspecific background staining. Slides were then incubated overnight with primary antibodies specific for CD4 (BD) and CD11c (Santa Cruz Biotechnology Inc.). Subsequently, the slides were incubated for 30 min at 20 1C with biotinylated secondary antibodies (Dianova). All samples were finally treated with streptavidin-horseradish peroxidase and stained with tyramide (Cy3), according to the manufacturer s instructions (PerkinElmer Life Sciences). Before examination, the nuclei were counterstained with Hoechst 33342 (Molecular Probes Inc.). Images are taken at 150 magnification. The pictures show a normal distribution of CD4 + and a higher number of CD11c + cells in the colon. 23 VOL.2 NO. 2007 NATURE PROTOCOLS

Figure 5 Isolation of LPMCs as described, and consequent staining of cells with propidium iodide (PI) and Annexin V to discriminate between dead and apoptotic cells. (a) Single staining with PI and analysis in FACS parameter FL3; (b) single staining with Annexin V conjugated with fluorescein isothiocyanate and analysis in FL1; (c) double staining with PI and Annexin V. The resulting cells show a necrotic amount of 2.5% and an apoptotic rate of 6.3%. This experiment represents a preparation under optimal conditions. 4 4 a 4 2.8 % b c 2.5% PI 3 3 3 1 1 1 11.7% 6.3% 0 0 0 0 1 3 4 0 1 3 4 0 1 3 4 FL1-H FL1-H FL1-H Annexin V Step 19 If the resulting cells should be used for proliferation assays or stimulation assays, some additional precautions should be taken. Cells should be washed with PBS before incubation. Use cell culture medium containing antibiotics such as 0 U ml 1 penicillin, 0 U ml 1 streptomycin, 5 mg ml 1 amphotericin B and mg ml 1 gentamycin. ANTICIPATED RESULTS The isolation of LPMCs from gut/colon tissue is time sensitive. The quicker the isolation, the better the status of the cells. For example, the analysis of CD4 + T cells from LPMC suspension needs fully expressed, and not digested, CD4 molecules on the surface of the living cells. The use of enzymes and EDTA alters the expression of T-cell surface antigens so that the subsequent FACS analysis may not reflect in detail the real percentage of cells in the suspension (Figs. 3,4). For this reason, it could be useful to test the effect of tryptic activity of the collagenase in pre-experiments with splenic cells in flow cytometric analysis. Additionally, the analysis of the cells should focus on the composition of the subpopulations. This gives an opportunity to compare various situations of the mucosa in different groups. Consequently, the cells are stained with antibodies for CD4 +, CD8 +, CD11b + and CD11c + to cover main cell populations. Normally, the subpopulations contain nearly 50% of the CD11c + cells (Fig. 3a). If the colon originates from mice suffering from colitis (e.g., transfer colitis with CD4 + CD25 + T cells), increased numbers of CD4 + T cells and less CD8 + T cells can be observed (Fig. 3b). Also, in immunohistochemically stained slides from cryosections of the colon, a higher number of CD11c + cells can be ascertained (Fig. 4). Finally, the resulting numbers of LPMC from isolation vary from about 0.5 3 6 cells per intestine. If isolating LPMCs from intestine originating from mice with intestinal inflammation, for example, Dextran Sulfate Sodium (DSS)-colitis model, increased numbers of LPMCs (1 7 6 ) can be ascertained. For the analysis of the quality of the resulting cells, a FACS staining with propidium iodide and Annexin V is necessary. This gives a good impression of the necrotic/apoptotic and living cells. Under optimum conditions, the percentage should be 2 5% necrotic/apoptotic and 5 % early apoptotic cells (Fig. 5). ACKNOWLEDGMENTS This work was supported by SFB 548. Published online at http://www.natureprotocols.com Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions 1. Schieferdecker, H.L., Ullrich, R., Hirseland, H. & Zeitz, M. T cell differentiation antigens on lymphocytes in the human intestinal lamina propria. J. Immunol. 149, 2816 2822 (1992). 2. Breese, E., Braegger, C.P., Corrigan, C.J., Walker-Smith, J.A. & MacDonald, T.T. Interleukin-2- and interferon-gamma-secreting T cells in normal and diseased human intestinal mucosa. Immunology 78, 127 131 (1993). 3. Guy-Grand, D., Cuenod-Jabri, B., Malassis-Seris, M., Selz, F. & Vassalli, P. Complexity of the mouse gut T cell immune system: identification of two distinct natural killer T cell intraepithelial lineages. Eur. J. Immunol. 26, 2248 2256 (1996). 4. 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