{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T17:05:13Z","timestamp":1780333513656,"version":"3.54.1"},"reference-count":18,"publisher":"Wiley","issue":"4","license":[{"start":{"date-parts":[[2026,4,22]],"date-time":"2026-04-22T00:00:00Z","timestamp":1776816000000},"content-version":"vor","delay-in-days":21,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/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>Recent discoveries linking cellular senescence to inflammaging and age\u2010associated metabolic diseases have intensified interest in defining the mechanisms that drive cellular senescence. Single\u2010cell transcriptomic studies have identified tissue\u2010resident macrophages, long\u2010lived and self\u2010renewing innate immune cells, as a major reservoir of senescent cells and key contributors to age\u2010associated chronic inflammation. Despite this progress, the upstream drivers that induce and stabilize macrophage senescence during aging and other comorbidities remain incompletely defined. Accumulating evidence now supports cholesterol loading as a\u00a0causal trigger of macrophage senescence, both in vitro and in vivo under hypercholesterolemic and high\u2010fat diet conditions. However, a critical gap in the field has been the lack of well\u2010defined experimental models to determine whether cholesterol\u2010loaded macrophages undergo true, stable cellular senescence, rather than a transient stress response that engages senescence\u2010associated pathways. Addressing this gap is essential for mechanistic studies and therapeutic targeting. Here, we describe the step\u2010by\u2010step methods to generate senescent macrophages using acetylated low\u2010density lipoprotein (Ac\u2010LDL), a model that we established and benchmarked against canonical DNA damage\u2010induced senescence macrophage models. We demonstrate that prolonged cholesterol loading drives a stable and reproducible senescent state in macrophages, characterized by persistent cell\u2010cycle arrest, expression of senescence\u2010associated secretory phenotype genes, and hallmark senescent phenotypes. This article provides a detailed protocol for generating and validating cholesterol\u2010induced senescent macrophages using Ac\u2010LDL, including complementary senescence assays to distinguish bona fide senescence from transient activation states. By standardizing experimental approaches to model macrophage senescence, these protocols will facilitate mechanistic studies and accelerate the development of targeted pharmacological strategies aimed at senescent immune cells in aging and metabolic disease. \u00a9 2026 Wiley Periodicals LLC.<\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol<\/jats:bold>\n                    : Ac\u2010LDL\u2010induced macrophage senescence in vitro\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Support Protocol<\/jats:bold>\n                    : Evaluation of cellular senescence in Ac\u2010LDL induced macrophage senescence\n                  <\/jats:p>","DOI":"10.1002\/cpz1.70355","type":"journal-article","created":{"date-parts":[[2026,4,22]],"date-time":"2026-04-22T10:51:16Z","timestamp":1776855076000},"source":"Crossref","is-referenced-by-count":1,"title":["Cholesterol\u2010Induced Senescent Macrophages and Evaluation of Cellular Senescence Phenotypes"],"prefix":"10.1002","volume":"6","author":[{"given":"Ivan A.","family":"Salladay\u2010Perez","sequence":"first","affiliation":[{"name":"Department of Microbiology Immunology and Molecular Genetics  Los Angeles California"},{"name":"Molecular Biology Institute University of California  Los Angeles California"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anthony J.","family":"Covarrubias","sequence":"additional","affiliation":[{"name":"Department of Microbiology Immunology and Molecular Genetics  Los Angeles California"},{"name":"Molecular Biology Institute University of California  Los Angeles California"},{"name":"David Geffen School of Medicine University of California  Los Angeles California"},{"name":"Broad Stem Cell Research Center University of California  Los Angeles California"},{"name":"Jonsson Comprehensive Cancer Center University of California  Los Angeles California"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,4,22]]},"reference":[{"key":"e_1_2_7_2_1","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.202010162"},{"key":"e_1_2_7_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41593\u2010020\u201000757\u20106"},{"key":"e_1_2_7_4_1","doi-asserted-by":"crossref","unstructured":"Chon J.(2025).L929 CONDITIONED MEDIUM (LCM) 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Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70355","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/cpz1.70355","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70355","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T16:20:18Z","timestamp":1780330818000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cpz1.70355"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4]]},"references-count":18,"journal-issue":{"issue":"4","published-print":{"date-parts":[[2026,4]]}},"alternative-id":["10.1002\/cpz1.70355"],"URL":"https:\/\/doi.org\/10.1002\/cpz1.70355","archive":["Portico"],"relation":{},"ISSN":["2691-1299","2691-1299"],"issn-type":[{"value":"2691-1299","type":"print"},{"value":"2691-1299","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4]]},"article-number":"e70355"}}