{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,7,30]],"date-time":"2025-07-30T09:45:50Z","timestamp":1753868750921,"version":"3.41.2"},"reference-count":24,"publisher":"Wiley","issue":"3","license":[{"start":{"date-parts":[[2024,3,26]],"date-time":"2024-03-26T00:00:00Z","timestamp":1711411200000},"content-version":"vor","delay-in-days":25,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2024,3]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Flow cytometry stands as the most employed high\u2010throughput single\u2010cell analysis technique, facilitating the profiling of remarkably diverse samples, such as blood, bone marrow and body fluids. In addition, it allows for the discrimination of diverse immune cell subsets, including infrequently encountered types like T regulatory cells and exhausted CD28<jats:sup>Null<\/jats:sup> T cells. However, analyzing rare immune cell subsets with conventional flow cytometry poses challenges stemming from factors like fluorophore overlap, compensation issues, and limited flexibility in fluorophore selection. Therefore, spectral flow cytometry offers advantages over traditional flow cytometry. It measures the full emission spectrum and then separates it to identify different fluorochromes. This enables the use of fluorochromes with significant overlap in a single test, allowing for the analysis of more protein markers. Following this, spectral technology employs precise calculations to separate individual fluorochromes, thereby enabling the detection and elimination of autofluorescent signals originating from cells within the entire emission spectrum. This capability is pivotal in achieving deep phenotyping of immune cells with the requisite sensitivity and resolution essential for monitoring the immune systems of patients with compromised immunity, such as cancer and autoimmune disorders. Additionally, it allows for the exploration of interactions between distinct immune subsets. In this context, we introduce an optimized protocol utilizing spectral flow cytometry for precise T\u2010cell characterization and differentiation, encompassing the assessment of their activation states. Furthermore, this protocol extends its applicability to the identification of less common circulating T\u2010cell populations, notably T\u2010regulatory and CD28<jats:sup>Null<\/jats:sup> T cells, following autofluorescence correction within the spectrum. This protocol provides a set of steps and reagents for the surface and intracellular staining of human T cells using whole peripheral blood. The spectral\u2010based design of this panel allows for its applicability to other spectral machines, providing a versatile and efficient tool for T\u2010cell analysis. \u00a9 2024 Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Achieving optimal staining through effective antibody titration<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Single\u2010cell staining<\/jats:p><jats:p><jats:bold>Basic Protocol 3<\/jats:bold>: Comprehensive panel staining post\u2010titration and spectral library integration<\/jats:p>","DOI":"10.1002\/cpz1.1020","type":"journal-article","created":{"date-parts":[[2024,3,26]],"date-time":"2024-03-26T10:30:15Z","timestamp":1711449015000},"source":"Crossref","is-referenced-by-count":0,"title":["Practical 10\u2010Color T\u2010Cell Panel for Phenotyping Diverse Populations Using Spectral Flow Cytometry: A Beginner's Guide"],"prefix":"10.1002","volume":"4","author":[{"given":"Sina","family":"Zargaran","sequence":"first","affiliation":[{"name":"Faculty of Dentistry University of Toronto  Toronto ON Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Morvarid","family":"Oveisi","sequence":"additional","affiliation":[{"name":"Faculty of Dentistry University of Toronto  Toronto ON Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chunxiang","family":"Sun","sequence":"additional","affiliation":[{"name":"Faculty of Dentistry University of Toronto  Toronto ON Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0184-8890","authenticated-orcid":false,"given":"Abdelahhad","family":"Barbour","sequence":"additional","affiliation":[{"name":"Faculty of Dentistry University of Toronto  Toronto ON Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael","family":"Glogauer","sequence":"additional","affiliation":[{"name":"Faculty of Dentistry University of Toronto  Toronto ON Canada"},{"name":"Department of Dental Oncology Princess Margaret Cancer Centre  Toronto Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2024,3,26]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/35105024"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1002\/9780471729259.mca02as00"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1007\/978\u20101\u201061737\u2010979\u20106_13"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1111\/imm.12506"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1161\/circresaha.111.261933"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1007\/978\u20101\u201061737\u2010979\u20106_17"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.3390\/cancers8030036"},{"key":"e_1_2_8_9_1","unstructured":"Group L. 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