Quantification of Cellular Senescence by SA-β-Gal Assay

To quantify the proportion of senescence associated β-galactosidase positive (SA-β-gal+) cells, the commercial kit “Cellular Senescence Assay” (EMD Millipore, Burlington, VT, USA, Catalog Number: KAA002) was used. The cells were stained according to supplemented manufacturer protocol with the following modification: at the final PBS washing step, the cell nuclei were stained with 1 μg/mL Hoechst 33342 (Molecular Probes, Eugene, OR, USA). Such modification significantly improves the quality of counting of β-galactosidase negative cells [24 (link),25 (link)]. The stained cells were visualized using Excitation/Emission Interference Filters (CKX-U: 340–380 nm/435–485 nm) on the inverted fluorescent microscope Olympus CKX 41 SF (Olympus, Tokyo, Japan) equipped with Infinity 3-1 (Lumenera Copr., Ottawa, Canada) camera and 20× objective. The proportions of SA-β-gal+ cells were counted manually (300 cells per each data point).