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However, the difficulty of multiparameter panel design combined with a lack of existing murine tools has prevented the comprehensive study of all major leukocyte phenotypes in a single assay. Herein, we present a 40\u2010color flow cytometry panel for deep immunophenotyping of murine lymphoid tissues, including the spleen, blood, Peyer's patches, inguinal lymph nodes, bone marrow, and thymus. This panel uses a robust set of surface markers capable of differentiating leukocyte subsets without the use of intracellular staining, thus allowing for the use of cells in downstream functional experiments or multiomic analyses. Our panel classifies T cells, B cells, natural killer cells, innate lymphoid cells, monocytes, macrophages, dendritic cells, basophils, neutrophils, eosinophils, progenitors, and their functional subsets by using a series of co\u2010stimulatory, checkpoint, activation, migration, and maturation markers. This tool has a multitude of systems immunology applications ranging from serial monitoring of circulating blood signatures to complex endpoint analysis, especially in pre\u2010clinical settings where treatments can modulate leukocyte abundance and\/or function. Ultimately, this 40\u2010color panel resolves a diverse array of immune cells on the axes of time, tissue, and treatment, filling the niche for a modern tool dedicated to murine immunophenotyping.<\/jats:p>","DOI":"10.1002\/cyto.a.24788","type":"journal-article","created":{"date-parts":[[2023,9,28]],"date-time":"2023-09-28T14:48:11Z","timestamp":1695912491000},"page":"839-850","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":35,"title":["<scp>OMIP\u2010095<\/scp>: 40\u2010Color spectral flow cytometry delineates all major leukocyte populations in murine lymphoid tissues"],"prefix":"10.1002","volume":"103","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4896-2853","authenticated-orcid":false,"given":"Aris J.","family":"Kare","sequence":"first","affiliation":[{"name":"Department of Bioengineering Stanford University  Stanford California USA"},{"name":"Department of Radiology Stanford University  Stanford California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3635-0435","authenticated-orcid":false,"given":"Lisa","family":"Nichols","sequence":"additional","affiliation":[{"name":"Stanford Shared FACS Facility Stanford University  Stanford California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ricardo","family":"Zermeno","sequence":"additional","affiliation":[{"name":"Stanford Shared FACS Facility Stanford University  Stanford California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5549-404X","authenticated-orcid":false,"given":"Marina N.","family":"Raie","sequence":"additional","affiliation":[{"name":"Department of Radiology Stanford University  Stanford California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0616-8280","authenticated-orcid":false,"given":"Spencer K.","family":"Tumbale","sequence":"additional","affiliation":[{"name":"Department of Radiology Stanford University  Stanford California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4976-9107","authenticated-orcid":false,"given":"Katherine W.","family":"Ferrara","sequence":"additional","affiliation":[{"name":"Department of Radiology Stanford University  Stanford California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2023,9,28]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2018.09.030"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2016.03.012"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.3389\/fmolb.2020.612801"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1126\/scisignal.aaa9303"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.51678"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.it.2016.12.007"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1038\/s43587-021-00148-x"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12979-021-00230-3"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1038\/ncb437"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1161\/circresaha.117.312513"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2016.08.015"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1038\/nn.4610"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2019.01315"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41590-021-01078-x"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.it.2012.02.010"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.22686"},{"key":"e_1_2_9_18_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2567.2008.02989.x"},{"key":"e_1_2_9_19_1","doi-asserted-by":"publisher","DOI":"10.1208\/s12248-021-00633-6"},{"key":"e_1_2_9_20_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.24211"},{"key":"e_1_2_9_21_1","doi-asserted-by":"crossref","unstructured":"LawrenceW VaradiG EntineG PodniesinskiE WallaceP.A comparison of avalanche photodiode and photomultiplier tube detectors for flow cytometry. 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