{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T05:11:21Z","timestamp":1788844281893,"version":"build-2803163510"},"reference-count":35,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,9,7]],"date-time":"2026-09-07T00:00:00Z","timestamp":1788739200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,7]],"date-time":"2026-09-07T00:00:00Z","timestamp":1788739200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/100008363","name":"Kr\u00e6ftens Bek\u00e6mpelse","doi-asserted-by":"publisher","award":["R302\u2010A17524"],"award-info":[{"award-number":["R302\u2010A17524"]}],"id":[{"id":"10.13039\/100008363","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Immunology"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    Monocytes play crucial roles in the pathobiology of a wide range of medical conditions, including infection, autoimmunity, cardiovascular disease, and malignancies. Studies using flow cytometry revealed the complexity of monocyte biology, including identification of three human monocyte subsets: classical (CD14\n                    <jats:sup>pos<\/jats:sup>\n                    CD16\n                    <jats:sup>neg<\/jats:sup>\n                    ), intermediate (CD14\n                    <jats:sup>pos<\/jats:sup>\n                    CD16\n                    <jats:sup>pos<\/jats:sup>\n                    ) and non\u2010classical (CD14\n                    <jats:sup>neg\/dim<\/jats:sup>\n                    CD16\n                    <jats:sup>pos<\/jats:sup>\n                    ) monocytes. Still, gating strategies used for identification of monocytes by flow cytometry vary substantially between studies, which may significantly impact results and hamper reproducibility. However, to our knowledge, no previous study has evaluated several different gating strategies for identification of the total circulating monocyte population in humans. Here, healthy donor peripheral blood mononuclear cells (PBMCs,\n                    <jats:italic>n<\/jats:italic>\n                    \u2009=\u200910) were analysed by flow cytometry to compare seven commonly used gating strategies for defining human monocytes: (1) negative selection (CD3\n                    <jats:sup>neg<\/jats:sup>\n                    CD19\n                    <jats:sup>neg<\/jats:sup>\n                    CD56\n                    <jats:sup>neg<\/jats:sup>\n                    CD66\n                    <jats:sup>neg<\/jats:sup>\n                    ), (2) FSC\u2010SSC, (3) CD14\n                    <jats:sup>pos<\/jats:sup>\n                    , (4) CD64\n                    <jats:sup>pos<\/jats:sup>\n                    , (5) HLA\u2010DR\n                    <jats:sup>pos<\/jats:sup>\n                    , (6) CD14\u2014HLA\u2010DR and (7) TLR2\n                    <jats:sup>pos<\/jats:sup>\n                    . We show that the different gating strategies significantly impacted the enumeration of non\u2010classical monocytes, but less so for classical and intermediate monocyte populations. Compared to negative selection gating (considered the reference standard), the FSC\u2010SSC\u2010based gating showed the highest degree of contamination by other cell types, whereas CD14\n                    <jats:sup>pos<\/jats:sup>\n                    , CD64\n                    <jats:sup>pos<\/jats:sup>\n                    , CD14\u2010HLA\u2010DR and HLA\u2010DR\n                    <jats:sup>pos<\/jats:sup>\n                    gating showed highest degree of exclusion of monocytes, particularly non\u2010classical monocytes. In comparison, TLR2\n                    <jats:sup>pos<\/jats:sup>\n                    gating showed both the least contamination and least erroneous exclusion of monocytes when compared to negative selection gating. These results based on \u2018manual gating\u2019 were supported by more objective t\u2010SNE and clustering analyses. In conclusion, TLR2\n                    <jats:sup>pos<\/jats:sup>\n                    gating and negative selection\u2010based gating displayed both minimal contamination and exclusion of true monocytes, with TLR2\n                    <jats:sup>pos<\/jats:sup>\n                    gating yielding the overall purest monocyte population. Thus, adapting to more uniform gating strategies for monocyte analysis, such as a TLR2 based gating strategy, may increase data quality and reproducibility between laboratories.\n                  <\/jats:p>","DOI":"10.1111\/imm.70195","type":"journal-article","created":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T04:41:15Z","timestamp":1788842475000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Evaluation of Methods for Identifying Human Monocytes by Flow Cytometry:\n                    <scp>TLR2<\/scp>\n                    May be an Optimal Marker"],"prefix":"10.1111","author":[{"ORCID":"https:\/\/orcid.org\/0009-0004-1115-3069","authenticated-orcid":false,"given":"Mie Wolff","family":"Kristensen","sequence":"first","affiliation":[{"name":"Department of Biomedicine Aarhus University  Aarhus Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katrine Skj\u00f8t","family":"Harbo","sequence":"additional","affiliation":[{"name":"Department of Biomedicine Aarhus University  Aarhus Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4140-6563","authenticated-orcid":false,"given":"Thomas","family":"Vorup\u2010Jensen","sequence":"additional","affiliation":[{"name":"Department of Biomedicine Aarhus University  Aarhus Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marianne","family":"Hokland","sequence":"additional","affiliation":[{"name":"Department of Biomedicine Aarhus University  Aarhus Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0820-7789","authenticated-orcid":false,"given":"Morten N\u00f8rgaard","family":"Andersen","sequence":"additional","affiliation":[{"name":"Department of Biomedicine Aarhus University  Aarhus Denmark"},{"name":"Department of Hematology Aarhus University Hospital  Aarhus Denmark"},{"name":"Department of Clinical Medicine Aarhus University  Aarhus Denmark"},{"name":"Department of Molecular Medicine (MOMA) Aarhus University Hospital  Aarhus Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,9,7]]},"reference":[{"key":"e_1_2_11_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/nri3671"},{"key":"e_1_2_11_3_1","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2015.00423"},{"key":"e_1_2_11_4_1","doi-asserted-by":"publisher","DOI":"10.3390\/v13091805"},{"key":"e_1_2_11_5_1","doi-asserted-by":"publisher","DOI":"10.1161\/ATVBAHA.118.311022"},{"key":"e_1_2_11_6_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms21165925"},{"key":"e_1_2_11_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrcardio.2009.228"},{"key":"e_1_2_11_8_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467\u2010024\u201045627\u2010y"},{"key":"e_1_2_11_9_1","doi-asserted-by":"publisher","DOI":"10.1182\/blood.V74.7.2527.2527"},{"key":"e_1_2_11_10_1","doi-asserted-by":"publisher","DOI":"10.1182\/blood\u20102010\u201002\u2010258558"},{"key":"e_1_2_11_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cellimm.2014.03.019"},{"key":"e_1_2_11_12_1","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2013.00023"},{"key":"e_1_2_11_13_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.20942"},{"key":"e_1_2_11_14_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.22995"},{"key":"e_1_2_11_15_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.24273"},{"key":"e_1_2_11_16_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.23098"},{"key":"e_1_2_11_17_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598\u2010019\u201043617\u20105"},{"key":"e_1_2_11_18_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms21176106"},{"key":"e_1_2_11_19_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.22104"},{"key":"e_1_2_11_20_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.24188"},{"key":"e_1_2_11_21_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.22455"},{"key":"e_1_2_11_22_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00109\u2010014\u20101200\u2010z"},{"key":"e_1_2_11_23_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.23331"},{"key":"e_1_2_11_24_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598\u2010020\u201061022\u20101"},{"key":"e_1_2_11_25_1","doi-asserted-by":"publisher","DOI":"10.1161\/ATVBAHA.117.309145"},{"key":"e_1_2_11_26_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.22703"},{"key":"e_1_2_11_27_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.3863"},{"key":"e_1_2_11_28_1","doi-asserted-by":"publisher","DOI":"10.2307\/2347485"},{"key":"e_1_2_11_29_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.b.22106"},{"key":"e_1_2_11_30_1","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2019.01761"},{"key":"e_1_2_11_31_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2017.11.001"},{"key":"e_1_2_11_32_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.24545"},{"key":"e_1_2_11_33_1","doi-asserted-by":"publisher","DOI":"10.1093\/ije\/dyac194"},{"key":"e_1_2_11_34_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41514\u2010024\u201000144\u20106"},{"key":"e_1_2_11_35_1","doi-asserted-by":"publisher","DOI":"10.1111\/ejh.13371"},{"key":"e_1_2_11_36_1","doi-asserted-by":"publisher","DOI":"10.1111\/imm.13125"}],"container-title":["Immunology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/imm.70195","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1111\/imm.70195","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/imm.70195","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T04:41:28Z","timestamp":1788842488000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1111\/imm.70195"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,7]]},"references-count":35,"alternative-id":["10.1111\/imm.70195"],"URL":"https:\/\/doi.org\/10.1111\/imm.70195","archive":["Portico"],"relation":{},"ISSN":["0019-2805","1365-2567"],"issn-type":[{"value":"0019-2805","type":"print"},{"value":"1365-2567","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,7]]},"assertion":[{"value":"2025-10-30","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication 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