In cell analyzer 6000

The slides were put in a 24-well plate, and then 2 × 10⁴ KYSE150 or KYSE450 cells were seeded in each well. After 16–18 h, cells were treated with various concentrations of azelnidipine for 24 h. The medium was then aspirated from the plate, and the cells were fixed with 4% paraformaldehyde in PBS for 30 min. The cells were washed three times with PBS and incubated primary antibody (p-ERK1/2 T202/Y204 and ERK1/2 1:50) overnight at 4°C. Next, slides were incubated with the fluorescent secondary antibody (1:50) for 1.5–2 h in the dark. After several washes, slides were stained with DAPI (1:10,000) for 5 min at 37°C. The images were captured and analyzed by using IN Cell Analyzer 6000 software.

Ercc1^−/− MEFs were passaged 5 times at 20% O₂ to induce senescence then seeded at 5000 cells per well in 96 well plates at least 6 h prior to treatment. Following the addition of drugs, the MEFs were incubated for 24–48 h at 20% O₂. Subsequently SA-β-gal activity was measured in three independent experiments, as previously described [49 (link)]. Briefly, cells were washed with PBS then 10 μM C₁₂FDG added in fresh culture medium and incubated for 2 h. Ten min prior to analysis, 2 μg/mL Hoechst dye was added. An IN Cell Analyzer 6000 was used to quantitate total number of viable cells (Hoechst⁺) and the number of senescent cells (C₁₂FDG⁺). All samples were analyzed in duplicate with 3–5 fields per well and reported as the mean ± S.D. Senotherapeutic activity was confirmed in human fibroblasts (IMR90). The cells were obtained from American Type Culture Collection (ATCC) and cultured in EMEM medium with 10% FBS and antibiotics. To induce senescence, the cells were treated for 24 h with 20 μM etoposide. Two days after etoposide removal, ~70% of the cells were SA-β-gal⁺. Cells were treated for 48 h with different concentrations of fisetin (1–15 μM) and the percentage of SA-β-gal⁺ cells was determined using C₁₂FDG, as described above.