Optimization of the SA-β-galactosidase assay for an enhanced senescence detection in H2O2-induced human umbilical vein endothelial cells (HUVECs)

Cellular senescence in endothelial cells (EC) plays a critical role in various age-related diseases, including cardiovascular disorders. Senescence-associated β-galactosidase (SA-β-gal) activity, detectable via cytochemical staining with X-gal, remains a widely accepted marker for identifying senescent cells. However, inconsistencies in senescence induction protocols hinder reproducibility and limit their utility in screening anti-senescence compounds. To date, no standardized in vitro conditions have been established for reliably inducing senescence in ECs. Herein, we aimed to utilize H2O2 as the primary inducer of cell senescence and investigated the effects of various concentrations, exposure time, chemical grade, and cell culture supplementation in the induction of senescence in human umbilical vein endothelial cells (HUVECs). In this study, we systematically evaluated a range of H₂O₂ concentration, exposure durations, chemical grades, and fetal bovine serum (FBS) supplementation to determine their effects on senescence induction. We quantified senescence by counting SA-β-gal-positive cells under a microscope after X-gal staining. Image-based cell counts, and statistical analyses were conducted to evaluate significance. We observed a threefold rise in SA-β-gal positivity after 24 hours of treatment with 0.5 µM H₂O₂ relative to the control. This indicates that optimal H2O2 concentration and exposure duration parameters significantly enhanced senescence. However, neither FBS supplementation nor H₂O₂ chemical grade influenced the outcomes. To conclude, this study highlights the need for an optimized senescence induction method to ensure reliable and interpretable results of SA-β-gal staining in HUVECs. Such improvements will facilitate future investigation of novel pharmacological agents with anti-senescence potential.