The comparison of methods for isolating immune cells from the intestinal lamina propria of mice and the immune cell landscape in DSS-induced colitis in mice
Abstract
Flow cytometry is a technology based on the detection of scattered light signals and fluorescent signals for multi-parameter, high-throughput, rapid analysis of individual cells or particles. It has broad application prospects in the biomedical field and is an indispensable technique, especially in immunological research. The preparation of a high-quality single-cell suspension is a key and often challenging step that directly affects the results of flow cytometry analysis. This study selected a primary tissue sample that is relatively difficult to prepare and commonly used in immunological research: The lamina propria of the mouse intestine. Three common enzymatic digestion methods were compared in terms of cell yield, viability, and immune cell markers. The results indicated that an enzymatic digestion method primarily using collagenase D is suitable for obtaining lymphocyte-derived immune cells from the mouse intestinal lamina propria, while enzymatic digestion methods primarily using LiberaseTM and DNase I are more suitable for obtaining myeloid-derived immune cells from the same tissue. Additionally, by combining a multi-parameter staining scheme, the analysis of T cells, macrophages, and dendritic cells within the intestinal immune cells was achieved. Furthermore, immunolabeling analysis was conducted on the commonly used colitis model, which was induced by dextran sulfate sodium, providing a reliable detection method for accurately analyzing immune cell populations in the mouse intestinal lamina propria.
References
1. Worbs T, Hammerschmidt SI, Förster R. Dendritic cell migration in health and disease. Nat Rev Immunol. 2017; 17(1): 30-48. doi: 10.1038/nri.2016.116
2. Kiner E, Willie E, Vijaykumar B, et al. Gut CD4+ T cell phenotypes are a continuum molded by microbes, not by TH archetypes. Nature Immunology. 2021; 22(2): 216–228.
3. Na YR, Stakenborg M, Seok SH, et al. Macrophages in intestinal inflammation and resolution: a potential therapeutic target in IBD. Nature Reviews Gastroenterology & Hepatology. 2019; 16(9): 531-543. doi: 10.1038/s41575-019-0172-4
4. Rosati E, Rios Martini G, Pogorelyy MV, et al. A novel unconventional T cell population enriched in Crohn’s disease. Gut. 2022; 71(11): 2194-2204. doi: 10.1136/gutjnl-2021-325373
5. Hang S, Paik D, Yao L, et al. Bile acid metabolites control TH17 and Treg cell differentiation. Nature. 2019; 576(7785): 143-148. doi: 10.1038/s41586-019-1785-z
6. Geremia A, Biancheri P, Allan P, et al. Innate and adaptive immunity in inflammatory bowel disease. Autoimmunity Reviews. 2014; 13(1): 3-10. doi: 10.1016/j.autrev.2013.06.004
7. Schulz O, Jaensson E, Persson EK, et al. Intestinal CD103+, but not CX3CR1+, antigen sampling cells migrate in lymph and serve classical dendritic cell functions. Journal of Experimental Medicine. 2009; 206(13): 3101-3114. doi: 10.1084/jem.20091925
8. Pabst O, Herbrand H, Friedrichsen M, et al. Adaptation of Solitary Intestinal Lymphoid Tissue in Response to Microbiota and Chemokine Receptor CCR7 Signaling. The Journal of Immunology. 2006; 177(10): 6824-6832. doi: 10.4049/jimmunol.177.10.6824
9. Bain CC, Bravo-Blas A, Scott CL, et al. Constant replenishment from circulating monocytes maintains the macrophage pool in the intestine of adult mice. Nature Immunology. 2014; 15(10): 929-937. doi: 10.1038/ni.2967
10. Bain CC, Scott CL, Uronen-Hansson H, et al. Resident and pro-inflammatory macrophages in the colon represent alternative context-dependent fates of the same Ly6Chi monocyte precursors. Mucosal Immunology. 2013; 6(3): 498-510. doi: 10.1038/mi.2012.89
11. Mitsialis V, Wall S, Liu P, et al. Single-Cell Analyses of Colon and Blood Reveal Distinct Immune Cell Signatures of Ulcerative Colitis and Crohn’s Disease. Gastroenterology. 2020; 159(2): 591-608.e10.
12. Anson ML. The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. Journal of General Physiology. 1938; 22(1): 79-89. doi: 10.1085/jgp.22.1.79
13. Harper E. Collagenases. Annual Review of Biochemistry. 1980; 49(1): 1063-1078. doi: 10.1146/annurev.bi.49.070180.005215
14. Wang K, Ren XW, Wang XY, et al. DNase I-Responsive Calixpyridinium-Mediated DNA Aggregation. Langmuir. 2019; 35(32): 10505-10511. doi: 10.1021/acs.langmuir.9b01116
15. Sexsmith E, Petersen WF. SKIN FERMENTS. Journal of Experimental Medicine. 1918; 27(2): 272-282. doi: 10.1084/jem.27.2.272
16. Abidi F, Aissaoui N, Chobert JM, et al. Neutral Serine Protease from Penicillium italicum. Purification, Biochemical Characterization, and Use for Antioxidative Peptide Preparation from Scorpaena notata Muscle. Applied Biochemistry and Biotechnology. 2014; 174(1): 186-205. doi: 10.1007/s12010-014-1052-6
17. Kamata Y, Taniguchi A, Yamamoto M, et al. Neutral Cysteine Protease Bleomycin Hydrolase Is Essential for the Breakdown of Deiminated Filaggrin into Amino Acids. Journal of Biological Chemistry. 2009; 284(19): 12829-12836. doi: 10.1074/jbc.m807908200
18. Hagiwara H, Miyazaki K, Matuo Y, et al. Purification and characterization of alkaline protease and neutral protease from chromatin of rats. Biochim Biophys Acta (BBA) - Enzymol. 1981; 660(1): 73–82.
19. Kim E, Tran M, Sun Y, et al. Isolation and analyses of lamina propria lymphocytes from mouse intestines. STAR Protocols. 2022; 3(2): 101366. doi: 10.1016/j.xpro.2022.101366
20. Wirtz S, Neufert C, Weigmann B, et al. Chemically induced mouse models of intestinal inflammation. Nature Protocols. 2007; 2(3): 541-546. doi: 10.1038/nprot.2007.41
21. Rindler K, Jonak C, Alkon N, et al. Single-cell RNA sequencing reveals markers of disease progression in primary cutaneous T-cell lymphoma. Molecular Cancer. 2021; 20(1). doi: 10.1186/s12943-021-01419-2
22. Kochenderfer JN, Yu Z, Frasheri D, et al. Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells. Blood. 2010; 116(19): 3875-3886. doi: 10.1182/blood-2010-01-265041
23. Rivollier A, He J, Kole A, et al. Inflammation switches the differentiation program of Ly6Chi monocytes from antiinflammatory macrophages to inflammatory dendritic cells in the colon. Journal of Experimental Medicine. 2012; 209(1): 139-155. doi: 10.1084/jem.20101387
24. Persson EK, Uronen-Hansson H, Semmrich M, et al. IRF4 Transcription-Factor-Dependent CD103+CD11b+ Dendritic Cells Drive Mucosal T Helper 17 Cell Differentiation. Immunity. 2013; 38(5): 958-969. doi: 10.1016/j.immuni.2013.03.009
25. Coombes JL, Siddiqui KRR, Arancibia-Cárcamo CV, et al. A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF-β– and retinoic acid–dependent mechanism. The Journal of Experimental Medicine. 2007; 204(8): 1757-1764. doi: 10.1084/jem.20070590
26. Esterházy D, Loschko J, London M, et al. Classical dendritic cells are required for dietary antigen–mediated induction of peripheral Treg cells and tolerance. Nature Immunology. 2016; 17(5): 545-555. doi: 10.1038/ni.3408
27. Welty NE, Staley C, Ghilardi N, et al. Intestinal lamina propria dendritic cells maintain T cell homeostasis but do not affect commensalism. Journal of Experimental Medicine. 2013; 210(10): 2011-2024. doi: 10.1084/jem.20130728
28. Cerovic V, Houston SA, Scott CL, et al. Intestinal CD103− dendritic cells migrate in lymph and prime effector T cells. Mucosal Immunology. 2013; 6(1): 104-113. doi: 10.1038/mi.2012.53
29. Tamoutounour S, Henri S, Lelouard H, et al. CD64 distinguishes macrophages from dendritic cells in the gut and reveals the Th1‐inducing role of mesenteric lymph node macrophages during colitis. European Journal of Immunology. 2012; 42(12): 3150-3166. doi: 10.1002/eji.201242847
30. Bond MD, Van Wart HE. Characterization of the individual collagenases from Clostridium histolyticum. Biochemistry. 1984; 23(13): 3085-3091. doi: 10.1021/bi00308a036
31. MacLennan JD, Mandl I, Howes EL. BACTERIAL DIGESTION OF COLLAGEN 1. Journal of Clinical Investigation. 1953; 32(12): 1317-1322. doi: 10.1172/jci102860
32. Matsushita O, Jung CM, Katayama S, et al. Gene Duplication and Multiplicity of Collagenases in Clostridium histolyticum. Journal of Bacteriology. 1999; 181(3): 923-933. doi: 10.1128/jb.181.3.923-933.1999
33. Fain JN. Isolation of free brown and white fat cells. Methods Enzymol. 1975; 35: 555–561.
34. Santerre K, Proulx S. Isolation efficiency of collagenase and EDTA for the culture of corneal endothelial cells. Mol Vis. 2022; 28: 331–339.
35. Berry MN, Friend DS. High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J Cell Biol. 1969; 43(3): 506–520.
36. Heyman E, Devriendt B, De Vlieghere E, et al. Evaluation of enzymatic protocols to optimize efficiency of bovine adipose tissue-derived mesenchymal stromal cell isolation. npj Science of Food. 2024; 8(1). doi: 10.1038/s41538-024-00313-7
37. Shingleton WD, Cawston TE, Hodges DJ, et al. Collagenase: a key enzyme in collagen turnover. Biochemistry and Cell Biology. 1996; 74(6): 759-775. doi: 10.1139/o96-083
38. Yamamoto T, Asano T, Mori A, et al. A rapid method for the separation of rat pancreatic islets from collagenase-digested pancreas using percoll. Endocrinologia Japonica. 1981; 28(5): 563-567. doi: 10.1507/endocrj1954.28.563
39. Dolmans MM, Michaux N, Camboni A, et al. Evaluation of Liberase, a purified enzyme blend, for the isolation of human primordial and primary ovarian follicles. Human Reproduction. 2005; 21(2): 413-420. doi: 10.1093/humrep/dei320
40. Chez MG, Dowling T, Patel PB, et al. Elevation of Tumor Necrosis Factor-Alpha in Cerebrospinal Fluid of Autistic Children. Pediatric Neurology. 2007; 36(6): 361-365. doi: 10.1016/j.pediatrneurol.2007.01.012
41. Śledź P, Kamiński R, Chruszcz M, et al. An experimental charge density of HEPES. Acta Crystallographica Section B Structural Science. 2010; 66(4): 482-492. doi: 10.1107/s0108768110023025
42. Suzuki T, Kubo K, Hori N, et al. Nonvolatile buffer coating of titanium to prevent its biological aging and for drug delivery. Biomaterials. 2010; 31(18): 4818-4828. doi: 10.1016/j.biomaterials.2010.02.061
43. Aso K, Kono M, Kanda M, et al. Itaconate ameliorates autoimmunity by modulating T cell imbalance via metabolic and epigenetic reprogramming. Nature Communications. 2023; 14(1). doi: 10.1038/s41467-023-36594-x
44. Peng M, Yin N, Chhangawala S, et al. Aerobic glycolysis promotes T helper 1 cell differentiation through an epigenetic mechanism. Science. 2016; 354(6311): 481-484. doi: 10.1126/science.aaf6284
45. Maloy KJ, Salaun L, Cahill R, et al. CD4+CD25+ TR Cells Suppress Innate Immune Pathology Through Cytokine-dependent Mechanisms. The Journal of Experimental Medicine. 2003; 197(1): 111-119. doi: 10.1084/jem.20021345
46. Reed MD, Yim YS, Wimmer RD, et al. IL-17a promotes sociability in mouse models of neurodevelopmental disorders. Nature. 2019; 577(7789): 249-253. doi: 10.1038/s41586-019-1843-6
47. Yang W, Cong Y. Exploring Colitis through Dynamic T Cell Adoptive Transfer Models. Inflammatory Bowel Diseases. 2023; 29(10): 1673-1680. doi: 10.1093/ibd/izad160
48. Macri C, Pang ES, Patton T, et al. Dendritic cell subsets. Seminars in Cell & Developmental Biology. 2018; 84: 11-21. doi: 10.1016/j.semcdb.2017.12.009
49. Zigmond E, Varol C, Farache J, et al. Ly6Chi Monocytes in the Inflamed Colon Give Rise to Proinflammatory Effector Cells and Migratory Antigen-Presenting Cells. Immunity. 2012; 37(6): 1076-1090. doi: 10.1016/j.immuni.2012.08.026
50. Siddiqui KRR, Laffont S, Powrie F. E-Cadherin Marks a Subset of Inflammatory Dendritic Cells that Promote T Cell-Mediated Colitis. Immunity. 2010; 32(4): 557-567. doi: 10.1016/j.immuni.2010.03.017
51. Schreiber HA, Loschko J, Karssemeijer RA, et al. Intestinal monocytes and macrophages are required for T cell polarization in response to Citrobacter rodentium. Journal of Experimental Medicine. 2013; 210(10): 2025-2039. doi: 10.1084/jem.20130903
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