{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T14:39:38Z","timestamp":1786977578936,"version":"build-2736575974"},"reference-count":25,"publisher":"Wiley","issue":"4","license":[{"start":{"date-parts":[[2026,4,17]],"date-time":"2026-04-17T00:00:00Z","timestamp":1776384000000},"content-version":"vor","delay-in-days":16,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"},{"start":{"date-parts":[[2026,4,1]],"date-time":"2026-04-01T00:00:00Z","timestamp":1775001600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2026,4]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    Telomere shortening is a hallmark of aging and immune dysfunction, offering a reliable indicator of cellular replicative history and biological age. Measuring telomere length in defined immune subsets provides critical insights into immune aging and disease\u2010associated remodeling. However, conventional methods often require prior cell sorting or compromise surface marker detection, limiting their ability to integrate telomere length with immunophenotyping. The advent of spectral flow cytometry enables highly multiplexed single\u2010sample analysis, overcoming many of these technical constraints. Here, we present a streamlined protocol that combines flow cytometry\u2013based fluorescence\n                    <jats:italic>in situ<\/jats:italic>\n                    hybridization (flow\u2010FISH) with spectral cytometry to measure relative telomere length (RTL) in peripheral blood mononuclear cells (PBMCs). This 18\u2010color panel simultaneously captures RTL across 15 immune subsets, alongside five functional and phenotypic markers reflecting activation, differentiation, senescence, and exhaustion. Our flow\u2010FISH method integrates telomere analysis and immune profiling into a single, reproducible workflow. Key optimizations include dual fixation to preserve membrane staining, use of the 1301 cell line as an internal telomere reference, and exclusion of dividing cells via cell cycle staining to ensure accurate RTL quantification. Validation in PBMCs from older adults demonstrated the method's ability to detect expected RTL decreases with T\u2010cell differentiation and senescence progression, confirming its utility for studies on immune aging, chronic infection, and other age\u2010related conditions. \u00a9 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 1<\/jats:bold>\n                    : Staining and flow cytometry for flow\u2010FISH to measure telomere length and immunophenotype in peripheral blood mononuclear cells (PBMCs)\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Support Protocol<\/jats:bold>\n                    : Preparation and cryopreservation of 1301 internal control cell line\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 2<\/jats:bold>\n                    : Data analysis and interpretation for telomere length and immunophenotype measurements in PBMCs\n                  <\/jats:p>","DOI":"10.1002\/cpz1.70359","type":"journal-article","created":{"date-parts":[[2026,4,17]],"date-time":"2026-04-17T15:31:08Z","timestamp":1776439868000},"source":"Crossref","is-referenced-by-count":1,"title":["Multiparametric Characterization of Telomere Length in T Cells by Flow\u2010FISH"],"prefix":"10.1002","volume":"6","author":[{"given":"Macedonia","family":"Trigueros","sequence":"first","affiliation":[{"name":"IrsiCaixa  Badalona Barcelona Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yacine","family":"Kharraz","sequence":"additional","affiliation":[{"name":"Application Department Cytek Biosciences Inc.  Fremont California"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hammad","family":"Alzayat","sequence":"additional","affiliation":[{"name":"Flow Cytometry Core Facility, Germans Trias i Pujol Research Institute  Badalona Spain"},{"name":"Current affiliation: Clinical Flow Cytometry Facility, Biomedical Diagnostic Center Hospital Clinic Barcelona  Barcelona Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juli\u00e0","family":"Blanco","sequence":"additional","affiliation":[{"name":"IrsiCaixa  Badalona Barcelona Spain"},{"name":"Centro de Investigaci\u00f3n Biom\u00e9dica en Red de Enfermedades Infecciosas CIBERINFEC  Madrid Spain"},{"name":"University of Vic\u2013Central University of Catalonia (UVic\u2010UCC)  Vic Catalonia Spain"},{"name":"Germans Trias i Pujol Research Institute Can Ruti Campus, UAB  Badalona Catalonia Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eugenia","family":"Negredo","sequence":"additional","affiliation":[{"name":"University of Vic\u2013Central University of Catalonia (UVic\u2010UCC)  Vic Catalonia Spain"},{"name":"Fight Against Infections Foundation Campus Can Ruti  Badalona Catalonia Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marco A.","family":"Fernandez","sequence":"additional","affiliation":[{"name":"Flow Cytometry Core Facility, Germans Trias i Pujol Research Institute  Badalona Spain"},{"name":"Current affiliation: Clinical Flow Cytometry Facility, Biomedical Diagnostic Center Hospital Clinic Barcelona  Barcelona Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marta","family":"Massanella","sequence":"additional","affiliation":[{"name":"IrsiCaixa  Badalona Barcelona Spain"},{"name":"Centro de Investigaci\u00f3n Biom\u00e9dica en Red de Enfermedades Infecciosas CIBERINFEC  Madrid Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,4,17]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.10053"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/ng1084"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1038\/350569a0"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0140\u20106736(03)12384\u20107"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1038\/365566a0"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-1771-7_4"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0113747"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.22257"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jim.2009.02.004"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.ado0431"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1098\/rsos.180492"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1101\/gad.1346005"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.1128\/mcb.10.2.518\u2010527.1990"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.1093\/hmg\/5.5.685"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.1038\/ng0198\u2010018"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.1182\/blood.2021013523"},{"key":"e_1_2_9_18_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrm2848"},{"key":"e_1_2_9_19_1","doi-asserted-by":"publisher","DOI":"10.3390\/cancers16132344"},{"key":"e_1_2_9_20_1","doi-asserted-by":"publisher","DOI":"10.1111\/mec.16145"},{"key":"e_1_2_9_21_1","doi-asserted-by":"publisher","DOI":"10.3389\/fonc.2023.1167848"},{"key":"e_1_2_9_22_1","doi-asserted-by":"publisher","DOI":"10.1159\/000540109"},{"key":"e_1_2_9_23_1","doi-asserted-by":"publisher","DOI":"10.1038\/labinvest.2016.100"},{"key":"e_1_2_9_24_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt0898\u2010743"},{"key":"e_1_2_9_25_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ebiom.2017.03.027"},{"key":"e_1_2_9_26_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.csbj.2024.04.011"}],"container-title":["Current 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