An In Vitro Model of Macrophage Senescence

We present a reproducible in vitro protocol for harvesting and culturing murine bone marrow-derived macrophages, with the added capability of freezing bone marrow cells at -80°C for scalability and long-term storage. To induce macrophage senescence, we developed a genotoxic stress-based method using either 10 Gy ionizing radiation or 500 nM doxorubicin treatment. The resulting senescent macrophages exhibit key hallmarks of cellular senescence, including irreversible cell cycle arrest, upregulation of senescence-associated markers (e.g., Cdkn1a), secretion of senescence-associated secretory phenotype (SASP) factors, morphological changes, and SA-β-galactosidase activity. This model serves as a valuable tool for investigating macrophage senescence, a relatively understudied senescent cell type, and provides mechanistic insights into the contribution of the innate immune system to aging and age-related diseases. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Mouse dissection and bone marrow harvest Basic Protocol 2: Thawing and plating cryopreserved murine bone marrow for macrophage differentiation Support Protocol 1: Flow cytometry validation of macrophage surface markers Support Protocol 2: Gene expression analysis via RT-qPCR Basic Protocol 3: Inducing senescence in macrophages using doxorubicin or irradiation Alternate Protocol: Maintenance and expansion of control macrophages Support Protocol 3: Brightfield microscopy and SA-β-galactosidase staining to assess senescence-associated morphology Support Protocol 4: Quantifying senescence and SASP marker expression by qPCR and/or western blot Support Protocol 5: Assessment of cell cycle arrest using EdU labeling with optional DNA content staining.

DNA damage; bone marrow‐derived macrophages; innate immune aging; macrophage differentiation; macrophage senescence.