{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T16:50:37Z","timestamp":1790009437725,"version":"4.0.1"},"reference-count":35,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2023,9,22]],"date-time":"2023-09-22T00:00:00Z","timestamp":1695340800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p>The three groups of helper innate lymphoid cells (ILCs), namely ILC1, ILC2 and ILC3, have been identified by flow cytometry by combinations of cell surface markers. Here, we review various ways ILCs are currently identified, focusing on potential problems and their solutions. The first step to identify all ILCs is to exclude other lymphocytes and myeloid cells by their lineage-specific markers (Lin). However, the Lin cocktail varies in various studies, and the definition of Lin- population containing ILCs is often ambiguous, resulting in contamination of Lin<jats:sup>+<\/jats:sup> cells, particularly T cells.<\/jats:p><\/jats:sec><jats:sec><jats:title>Method<\/jats:title><jats:p>We have designed combinations of cell surface markers to identify ILC populations in various tissues of B6 mice by flow cytometry. To minimize T cell contamination, TCR\/CD3\u03f5 antibodies were used separately from the Lin cocktail. ILCs identified by surface markers are confirmed by the expression of the transcription factors GATA3, ROR\u03b3t, T-bet and Eomes.<\/jats:p><\/jats:sec><jats:sec><jats:title>Result<\/jats:title><jats:p>ILC1s in the B6 mouse liver are identified by Lin-NKp46<jats:sup>+<\/jats:sup>NK1.1<jats:sup>+<\/jats:sup>TCR\/CD3\u03f5<jats:sup>\u2212<\/jats:sup>CD49a<jats:sup>+<\/jats:sup>CD49b<jats:sup>\u2212<\/jats:sup>. However, defining ILC1s in other tissues remains a challenge. ILC2s in the lung are identified by Lin<jats:sup>\u2212<\/jats:sup>TCR\/CD3\u03f5<jats:sup>\u2212<\/jats:sup> Thy1<jats:sup>+<\/jats:sup>CD127<jats:sup>+<\/jats:sup>ST2<jats:sup>+<\/jats:sup> whereas ILC2s in the small intestine and liver are identified by Lin<jats:sup>\u2212<\/jats:sup>TCR\/CD3\u03f5<jats:sup>\u2212<\/jats:sup>Thy1<jats:sup>+<\/jats:sup>GATA3<jats:sup>+<\/jats:sup>ROR\u03b3t<jats:sup>\u2212<\/jats:sup>. ILC3s in B6 mouse spleen, liver, lung and small intestine are identified by Lin<jats:sup>\u2212<\/jats:sup>TCR\/CD3\u03f5<jats:sup>\u2212<\/jats:sup> Thy1<jats:sup>+<\/jats:sup>CD127<jats:sup>+<\/jats:sup>ROR\u03b3t<jats:sup>+<\/jats:sup>.<\/jats:p><\/jats:sec><jats:sec><jats:title>Discussion<\/jats:title><jats:p>The ILC population is heterogeneous and the strategies to identify ILCs have to be designed for each ILC population and tissue. Excluding T cells in all cases is crucial, and a combination of transcription factors GATA3, ROR\u03b3t, T-bet, and Eomes should be used to identify ILCs. Using CD3\u03f5\/TCRs in a different fluorochrome not in Lin cocktail minimizes contamination of T cells specifically identify individual ILC populations in various tissues.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fimmu.2023.1198310","type":"journal-article","created":{"date-parts":[[2023,9,22]],"date-time":"2023-09-22T09:39:56Z","timestamp":1695375596000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":22,"title":["Flow cytometric analysis of innate lymphoid cells: challenges and solutions"],"prefix":"10.3389","volume":"14","author":[{"given":"Mona","family":"Sadeghalvad","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Davit","family":"Khijakadze","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mona","family":"Orangi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fumio","family":"Takei","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2023,9,22]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2021.671966","article-title":"The fate of activated group 2 innate lymphoid cells","volume":"12","author":"Math\u00e4","year":"2021","journal-title":"Front Immunol"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4939-8549-4_21","article-title":"Isolation and identification of innate lymphoid cells (ILCs) for immunotoxicity testing","volume":"1803","author":"Tait Wojno","year":"2018","journal-title":"Immunotoxicity Testing: Methods and Protocols"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2008-10-187179","article-title":"Maturation of mouse NK cells is a 4-stage developmental program","volume":"113","author":"Chiossone","year":"2009","journal-title":"Blood"},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.1038\/s41577-022-00704-5","article-title":"Heterogeneity of type 2 innate lymphoid cells","volume":"22","author":"Spits","year":"2022","journal-title":"Nat Rev Immunol"},{"key":"B5","doi-asserted-by":"publisher","first-page":"100812","DOI":"10.1016\/j.xcrm.2022.100812","article-title":"Tissue-specific transcriptional profiles and heterogeneity of natural killer cells and group 1 innate lymphoid cells","volume":"3","author":"Lopes","year":"2022","journal-title":"Cell Rep Med"},{"key":"B6","doi-asserted-by":"publisher","first-page":"jem.20182293","DOI":"10.1084\/jem.20182293","article-title":"Single-cell analysis of ROR\u03b1 tracer mouse lung reveals ILC progenitors and effector ILC2 subsets","volume":"217","author":"Ghaedi","year":"2020","journal-title":"J Exp Med"},{"key":"B7","doi-asserted-by":"publisher","first-page":"83","DOI":"10.1016\/j.immuni.2019.12.001","article-title":"Differential expression of the transcription factor GATA3 specifies lineage and functions of innate lymphoid cells","volume":"52","author":"Zhong","year":"2020","journal-title":"Immunity"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2020.563414","article-title":"Optimized protocol for characterization of mouse gut innate lymphoid cells","volume":"11","author":"Valle-Noguera","year":"2020","journal-title":"Front Immunol"},{"key":"B9","doi-asserted-by":"publisher","first-page":"48","DOI":"10.1016\/j.coi.2020.08.009","article-title":"Natural killers or ILC1s? 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