Senescence Detection Using Reflected Light in Adipose Stromal Vascular Fraction

Cellular senescence is a stress-induced state characterized by permanent cell-cycle arrest and the development of a distinctive secretory profile that impacts tissue function and contributes to aging and metabolic disease. Senescence-associated β-galactosidase (SA-β-gal) activity is widely used as a marker of senescent cells; however, conventional SA-β-gal assays often rely on subjective visual assessment and provide limited quantitative information. These limitations are particularly evident in primary human cell populations such as the stromal vascular fraction (SVF) derived from adipose tissue, which contains a heterogeneous mixture of preadipocytes, immune cells, and endothelial cells. Here, we present an optimized reflected light confocal microscopy approach for high-resolution, quantitative detection of SA-β-gal activity in human SVF cells. This protocol enables objective single-cell analysis of SA-β-gal activity, allows simultaneous immunocytochemistry for multiplexed detection of additional senescence or lineage markers, and incorporates pH-matched controls. By combining these features, this method provides a sensitive, reproducible, and quantitative approach to studying cellular senescence in heterogeneous primary human cell populations. It offers an improved alternative to conventional SA-β-gal staining.