Cholesterol‐Induced Senescent Macrophages and Evaluation of Cellular Senescence Phenotypes

Recent discoveries linking cellular senescence to inflammaging and age-associated metabolic diseases have intensified interest in defining the mechanisms that drive cellular senescence. Single-cell transcriptomic studies have identified tissue-resident macrophages, long-lived and self-renewing innate immune cells, as a major reservoir of senescent cells and key contributors to age-associated 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 causal trigger of macrophage senescence, both in vitro and in vivo under hypercholesterolemic and high-fat diet conditions. However, a critical gap in the field has been the lack of well-defined experimental models to determine whether cholesterol-loaded macrophages undergo true, stable cellular senescence, rather than a transient stress response that engages senescence-associated pathways. Addressing this gap is essential for mechanistic studies and therapeutic targeting. Here, we describe the step-by-step methods to generate senescent macrophages using acetylated low-density lipoprotein (Ac-LDL), a model that we established and benchmarked against canonical DNA damage-induced senescence macrophage models. We demonstrate that prolonged cholesterol loading drives a stable and reproducible senescent state in macrophages, characterized by persistent cell-cycle arrest, expression of senescence-associated secretory phenotype genes, and hallmark senescent phenotypes. This article provides a detailed protocol for generating and validating cholesterol-induced senescent macrophages using Ac-LDL, 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. © 2026 Wiley Periodicals LLC. Basic Protocol: Ac-LDL-induced macrophage senescence in vitro Support Protocol: Evaluation of cellular senescence in Ac-LDL induced macrophage senescence.