{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T13:01:43Z","timestamp":1785416503863,"version":"3.56.0"},"reference-count":44,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2026,6,12]],"date-time":"2026-06-12T00:00:00Z","timestamp":1781222400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004329","name":"Slovenian Research and Innovation Agency","doi-asserted-by":"publisher","award":["P3\u20130003"],"award-info":[{"award-number":["P3\u20130003"]}],"id":[{"id":"10.13039\/501100004329","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004329","name":"Slovenian Research and Innovation Agency","doi-asserted-by":"publisher","award":["Z3\u201350112"],"award-info":[{"award-number":["Z3\u201350112"]}],"id":[{"id":"10.13039\/501100004329","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004329","name":"Slovenian Research and Innovation Agency","doi-asserted-by":"publisher","award":["I0-0059"],"award-info":[{"award-number":["I0-0059"]}],"id":[{"id":"10.13039\/501100004329","id-type":"DOI","asserted-by":"publisher"}]},{"award":["P3\u20130003"],"award-info":[{"award-number":["P3\u20130003"]}],"id":[{"id":"https:\/\/ror.org\/059bp8k51","id-type":"ROR","asserted-by":"publisher"}]},{"award":["Z3\u201350112"],"award-info":[{"award-number":["Z3\u201350112"]}],"id":[{"id":"https:\/\/ror.org\/059bp8k51","id-type":"ROR","asserted-by":"publisher"}]},{"award":["I0-0059"],"award-info":[{"award-number":["I0-0059"]}],"id":[{"id":"https:\/\/ror.org\/059bp8k51","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["MPs"],"abstract":"<jats:p>Lymph nodes are central hubs of immune regulation and coordination, serving as primary sites for antigen presentation, lymphocyte activation, and the orchestration of adaptive immune responses. The composition and activation state of lymph node-resident immune cells critically shape both local and systemic immunity. Comprehensive immunophenotyping of these populations is therefore essential for understanding immune organization and functional heterogeneity. Here, we present an optimized protocol for the characterization of mouse lymph node-associated immune populations using 14-color multiparametric flow cytometry. The method combines lymph node isolation based on anatomical landmarks with mechanical dissociation and enzymatic digestion to generate high-quality single-cell suspensions suitable for downstream analysis. Furthermore, the described flow cytometry panel and gating strategy enable reliable identification and quantification of major lymphoid subsets, including helper CD4+ and cytotoxic CD8+ T cells with their differentiation states, as well as natural killer (NK) cells across distinct maturation stages. Although optimized for assessing lymphocyte maturation after lipopolysaccharide (LPS) challenge, the protocol serves as a reproducible platform for broad immunophenotyping of T and NK cell subsets in mouse lymphoid tissues under experimental conditions.<\/jats:p>","DOI":"10.3390\/mps9030097","type":"journal-article","created":{"date-parts":[[2026,6,18]],"date-time":"2026-06-18T02:10:16Z","timestamp":1781748616000},"page":"97","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Multiparametric Flow Cytometry Panel for Characterization of Mouse T Cell Differentiation and NK Cell Maturation Following Inflammatory Challenge"],"prefix":"10.3390","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6468-8287","authenticated-orcid":false,"given":"Tim","family":"Bozic","sequence":"first","affiliation":[{"name":"Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska cesta 2, SI-1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8674-702X","authenticated-orcid":false,"given":"Bostjan","family":"Markelc","sequence":"additional","affiliation":[{"name":"Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska cesta 2, SI-1000 Ljubljana, Slovenia"},{"name":"Biotechnical Faculty, University of Ljubljana, Jamnikarjeva ulica 101, SI-1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0538-7841","authenticated-orcid":false,"given":"Simona","family":"Kranjc Brezar","sequence":"additional","affiliation":[{"name":"Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska cesta 2, SI-1000 Ljubljana, Slovenia"},{"name":"Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ziva","family":"Pisljar","sequence":"additional","affiliation":[{"name":"Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska cesta 2, SI-1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5883-1673","authenticated-orcid":false,"given":"Tanja","family":"Jesenko","sequence":"additional","affiliation":[{"name":"Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska cesta 2, SI-1000 Ljubljana, Slovenia"},{"name":"Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1418-1928","authenticated-orcid":false,"given":"Maja","family":"Cemazar","sequence":"additional","affiliation":[{"name":"Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska cesta 2, SI-1000 Ljubljana, Slovenia"},{"name":"Faculty of Health Sciences, University of Primorska, Polje 42, SI-6310 Izola, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Xiang, M., Grosso, R.A., Takeda, A., Pan, J., Bekkhus, T., Brulois, K., Dermadi, D., Nordling, S., Vanlandewijck, M., and Jalkanen, S. (2020). A Single-Cell Transcriptional Roadmap of the Mouse and Human Lymph Node Lymphatic Vasculature. Front. Cardiovasc. Med., 7.","DOI":"10.3389\/fcvm.2020.00052"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1016\/j.it.2014.11.003","article-title":"Stromal infrastructure of the lymph node and coordination of immunity","volume":"36","author":"Chang","year":"2015","journal-title":"Trends Immunol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"441","DOI":"10.1016\/j.cell.2020.11.028","article-title":"Lymph nodes are innervated by a unique population of sensory neurons with immunomodulatory potential","volume":"184","author":"Huang","year":"2021","journal-title":"Cell"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"762","DOI":"10.1038\/nri3298","article-title":"HEVs, lymphatics and homeostatic immune cell trafficking in lymph nodes","volume":"12","author":"Girard","year":"2012","journal-title":"Nat. Rev. Immunol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"e714","DOI":"10.1002\/mco2.714","article-title":"The interaction of innate immune and adaptive immune system","volume":"5","author":"Wang","year":"2024","journal-title":"MedComm"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"375","DOI":"10.1038\/s41577-020-0285-6","article-title":"Defining trained immunity and its role in health and disease","volume":"20","author":"Netea","year":"2020","journal-title":"Nat. Rev. Immunol."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Moi, D., Zeng, B., Minnie, S.A., Bhatt, R., Wood, J., Sester, D.P., Mazzieri, R., and Dolcetti, R. (2023). Multiparametric flow cytometry to characterize vaccine-induced polyfunctional T cell responses and T cell\/NK cell exhaustion and memory phenotypes in mouse immuno-oncology models. Front. Immunol., 14.","DOI":"10.3389\/fimmu.2023.1127896"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"551","DOI":"10.1002\/eji.1830220238","article-title":"CD45 modulates T cell receptor\/CD3-induced activation of human thymocytes via regulation of tyrosine phosphorylation","volume":"22","author":"Turka","year":"1992","journal-title":"Eur. J. Immunol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"508","DOI":"10.4161\/cib.3.6.13495","article-title":"Multifaceted regulation of T cells by CD44","volume":"3","author":"Baaten","year":"2010","journal-title":"Commun. Integr. Biol."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Yang, S., Liu, F., Wang, Q.J., Rosenberg, S.A., and Morgan, R.A. (2011). The shedding of CD62L (L-selectin) regulates the acquisition of lytic activity in human tumor reactive T lymphocytes. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0022560"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"e2103730118.","DOI":"10.1073\/pnas.2103730118","article-title":"Critical role of the CD44 low CD62L low CD8 + T cell subset in restoring antitumor immunity in aged mice","volume":"118","author":"Nakajima","year":"2021","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1186\/s40425-017-0235-4","article-title":"Differential phenotypes of memory CD4 and CD8 T cells in the spleen and peripheral tissues following immunostimulatory therapy","volume":"5","author":"Sckisel","year":"2017","journal-title":"J. Immunother. Cancer"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Abel, A.M., Yang, C., Thakar, M.S., and Malarkannan, S. (2018). Natural killer cells: Development, maturation, and clinical utilization. Front. Immunol., 9.","DOI":"10.3389\/fimmu.2018.01869"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Goh, W., and Huntington, N.D. (2017). Regulation of murine natural killer cell development. Front. Immunol., 8.","DOI":"10.3389\/fimmu.2017.00130"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"573","DOI":"10.1016\/j.it.2013.07.005","article-title":"Location and cellular stages of natural killer cell development","volume":"34","author":"Yu","year":"2013","journal-title":"Trends Immunol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"658","DOI":"10.1038\/nri3065","article-title":"Organ-specific features of natural killer cells","volume":"11","author":"Shi","year":"2011","journal-title":"Nat. Rev. Immunol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1016\/j.clim.2014.10.005","article-title":"BCG-induced trained immunity in NK cells: Role for non-specific protection to infection","volume":"155","author":"Kleinnijenhuis","year":"2014","journal-title":"Clin. Immunol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1186\/s13045-022-01243-1","article-title":"A four-stage model for murine natural killer cell development in vivo","volume":"15","author":"Ma","year":"2022","journal-title":"J. Hematol. Oncol."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Wu, Y., Tian, Z., and Wei, H. (2017). Developmental and functional control of natural killer cells by cytokines. Front. Immunol., 8.","DOI":"10.3389\/fimmu.2017.00930"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"4526","DOI":"10.1073\/pnas.072065999","article-title":"Early expression of triggering receptors and regulatory role of 2B4 in human natural killer cell precursors undergoing in vitro differentiation","volume":"99","author":"Sivori","year":"2002","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"712","DOI":"10.1021\/pr800747c","article-title":"Natural Cytotoxicity Receptors NKp30, NKp44 and NKp46 Bind to Different Heparan Sulfate\/Heparin Sequences","volume":"8","author":"Hecht","year":"2009","journal-title":"J. Proteome Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e13435","DOI":"10.1111\/imr.13435","article-title":"Recognition of Self and Viral Ligands by NK Cell Receptors","volume":"329","author":"Mariuzza","year":"2025","journal-title":"Immunol. Rev."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"3384","DOI":"10.1073\/pnas.0609692104","article-title":"Identification, activation, and selective in vivo ablation of mouse NK cells via NKp46","volume":"104","author":"Walzer","year":"2007","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"683","DOI":"10.1038\/s41467-024-44789-z","article-title":"Rapid functional impairment of natural killer cells following tumor entry limits anti-tumor immunity","volume":"15","author":"Dean","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"523","DOI":"10.1038\/ni796","article-title":"In vivo developmental stages in murine natural killer cell maturation","volume":"3","author":"Kim","year":"2002","journal-title":"Nat. Immunol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1802","DOI":"10.1093\/jimmun\/vkaf105","article-title":"Ly49G, but not Ly49C\/I, is dispensable for diverse antigen-specific memory NK cell responses in H-2d and H-2b mice","volume":"214","author":"Gamage","year":"2025","journal-title":"J. Immunol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"900","DOI":"10.4049\/jimmunol.166.2.900","article-title":"CD11b Expression Identifies CD8+CD28+ T Lymphocytes with Phenotype and Function of Both Naive\/Memory and Effector Cells1","volume":"166","author":"Fiorentini","year":"2001","journal-title":"J. Immunol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"799","DOI":"10.1038\/s43587-025-00855-9","article-title":"KLRG1 identifies regulatory T cells with mitochondrial alterations that accumulate with aging","volume":"5","author":"Carrasco","year":"2025","journal-title":"Nat. Aging"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e184843","DOI":"10.1172\/jci.insight.184843","article-title":"CD154:CD11b blockade enhances CD8+ T cell differentiation during infection but not transplantation","volume":"10","author":"Alexander","year":"2025","journal-title":"JCI Insight"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1186\/s12964-024-01714-7","article-title":"The role of KLRG1: A novel biomarker and new therapeutic target","volume":"22","author":"Zhang","year":"2024","journal-title":"Cell Commun. Signal."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2514","DOI":"10.1158\/1078-0432.CCR-23-3504","article-title":"KLRG1 Cell Depletion as a Novel Therapeutic Strategy in Patients with Mature T-Cell Lymphoma Subtypes","volume":"30","author":"Assatova","year":"2024","journal-title":"Clin. Cancer Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"eadj8356","DOI":"10.1126\/sciimmunol.adj8356","article-title":"Circulating KLRG1+ long-lived effector memory T cells retain the flexibility to become tissue resident","volume":"9","author":"Lucas","year":"2024","journal-title":"Sci. Immunol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"e005061","DOI":"10.1136\/jitc-2022-005061","article-title":"N-cadherin protects oral cancer cells from NK cell killing in the circulation by inducing NK cell functional exhaustion via the KLRG1 receptor","volume":"10","author":"Lou","year":"2022","journal-title":"J. Immunother. Cancer"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1007\/s00262-025-04059-3","article-title":"KLRG1 expression induces functional exhaustion of NK cells in colorectal cancer patients","volume":"74","author":"Xu","year":"2025","journal-title":"Cancer Immunol. Immunother."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"101202","DOI":"10.1016\/j.xcrm.2023.101202","article-title":"KLRG1 expression on natural killer cells is associated with HIV persistence, and its targeting promotes the reduction of the viral reservoir","volume":"4","author":"Perea","year":"2023","journal-title":"Cell Rep. Med."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"3933","DOI":"10.1016\/j.celrep.2019.11.043","article-title":"H3K4me1 Supports Memory-like NK Cells Induced by Systemic Inflammation","volume":"29","author":"Rasid","year":"2019","journal-title":"Cell Rep."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1889","DOI":"10.1111\/jgh.15375","article-title":"Lipopolysaccharide preconditioning reduces liver metastasis of Colon26 cells by enhancing antitumor activity of natural killer cells and natural killer T cells in murine liver","volume":"36","author":"Nishikawa","year":"2021","journal-title":"J. Gastroenterol. Hepatol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"3612","DOI":"10.1038\/s41467-025-58931-y","article-title":"Inflammatory conditions shape phenotypic and functional characteristics of lung-resident memory T cells in mice","volume":"16","author":"Schmidt","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"118107","DOI":"10.1016\/j.biopha.2025.118107","article-title":"Partial versus whole tumor-volume irradiation in combination with immunotherapy: Comparable outcomes in immunosuppressed mouse models of oral squamous cell carcinoma","volume":"187","author":"Pisljar","year":"2025","journal-title":"Biomed. Pharmacother."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1177\/0023677217724823","article-title":"PREPARE: Guidelines for planning animal research and testing","volume":"52","author":"Smith","year":"2018","journal-title":"Lab. Anim."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1016\/j.jim.2006.01.022","article-title":"Anatomy and nomenclature of murine lymph nodes: Descriptive study and nomenclatory standardization in BALB\/cAnNCrl mice","volume":"312","author":"Derore","year":"2006","journal-title":"J. Immunol. Methods"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"2708","DOI":"10.1002\/eji.202170126","article-title":"Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)","volume":"51","author":"Cossarizza","year":"2021","journal-title":"Eur. J. Immunol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"e70","DOI":"10.1002\/cpcy.70","article-title":"Panel Design and Optimization for High-Dimensional Immunophenotyping Assays Using Spectral Flow Cytometry","volume":"92","author":"Pellefigues","year":"2020","journal-title":"Curr. Protoc. Cytom."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1111\/imm.12706","article-title":"Isolation and high-dimensional phenotyping of gastrointestinal immune cells","volume":"151","author":"David","year":"2017","journal-title":"Immunology"}],"container-title":["Methods and Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2409-9279\/9\/3\/97\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,18]],"date-time":"2026-06-18T02:28:04Z","timestamp":1781749684000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2409-9279\/9\/3\/97"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,12]]},"references-count":44,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2026,6]]}},"alternative-id":["mps9030097"],"URL":"https:\/\/doi.org\/10.3390\/mps9030097","relation":{},"ISSN":["2409-9279"],"issn-type":[{"value":"2409-9279","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,12]]}}}