{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,27]],"date-time":"2026-06-27T09:11:22Z","timestamp":1782551482535,"version":"3.54.5"},"reference-count":26,"publisher":"Wiley","issue":"7","license":[{"start":{"date-parts":[[2023,7,10]],"date-time":"2023-07-10T00:00:00Z","timestamp":1688947200000},"content-version":"vor","delay-in-days":9,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2023,7]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>This article discusses methods to assess invariant natural killer T (iNKT) cell subsets isolated from the thymus, as well as the spleen, the liver, and the lung. iNKT cells can be subdivided in distinct, functional subsets based on the transcription factors they express and the cytokines they produce to regulate the immune response. Basic Protocol 1 focuses on characterizing murine iNKT subsets <jats:italic>ex vivo<\/jats:italic> by flow cytometry by evaluating the expression of lineage\u2010specifying transcription factors such as PLZF and ROR\u03b3t. The Alternate Protocol describes a detailed approach to define subsets based on expression of surface markers. This approach can be very useful for maintaining the subsets alive, without fixing them, in order to isolate them for downstream molecular assays such as DNA\/RNA isolation, genome\u2010wide analysis to assess gene expression (such as RNA\u2010seq), assessment of chromatin accessibility (for instance, by ATAC\u2010seq), and assessment of DNA methylation by whole\u2010genome bisulfite sequencing. Basic Protocol 2 describes the functional characterization of iNKT cells, which are activated <jats:italic>in vitro<\/jats:italic> with PMA and ionomycin for a short period of time and subsequently stained and characterized for production of cytokines, such as IFN\u03b3 and IL\u20104, by flow cytometry. Basic Protocol 3 describes the process of activating iNKT cells <jats:italic>in vivo<\/jats:italic> using \u03b1\u2010galactosyl\u2010ceramide, a lipid that can be recognized specifically by iNKT cells, allowing assessment of their functionality <jats:italic>in vivo<\/jats:italic>. Cells are then isolated and directly stained for cytokine secretion. \u00a9 2023 Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Identifying iNKT cell subsets based on transcription factor expression by flow cytometry<\/jats:p><jats:p><jats:bold>Alternate Protocol<\/jats:bold>: Identifying iNKT cell subsets based on surface marker expression by flow cytometry<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: iNKT cell functional characterization based on <jats:italic>in vitro<\/jats:italic> activation and assessment of cytokine secretion<\/jats:p><jats:p><jats:bold>Basic Protocol 3<\/jats:bold>: iNKT cell <jats:italic>in vivo<\/jats:italic> activation and assessment of cytokine secretion by flow cytometry<\/jats:p>","DOI":"10.1002\/cpz1.838","type":"journal-article","created":{"date-parts":[[2023,7,10]],"date-time":"2023-07-10T18:29:35Z","timestamp":1689013775000},"source":"Crossref","is-referenced-by-count":6,"title":["Defining iNKT Cell Subsets and Their Function by Flow Cytometry"],"prefix":"10.1002","volume":"3","author":[{"given":"Marianthi","family":"Gioulbasani","sequence":"first","affiliation":[{"name":"Lineberger Comprehensive Cancer Center University of North Carolina at Chapel Hill  Chapel Hill North Carolina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ageliki","family":"Tsagaratou","sequence":"additional","affiliation":[{"name":"Lineberger Comprehensive Cancer Center University of North Carolina at Chapel Hill  Chapel Hill North Carolina"},{"name":"Department of Genetics University of North Carolina at Chapel Hill  Chapel Hill North Carolina"},{"name":"Department of Microbiology and Immunology University of North Carolina  Chapel Hill North 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