2.9. In Situ Staining of Senescence-Associated β-Galactosidase Activity, Autofluorescence Analysis, and Detection of Apoptosis

To detect senescence-associated β-galactosidase activity, ME180, MS751, and SCC152 cells (2.5 × 10³) were seeded into 24-well plates and the next day cells were treated for 72 h with test compounds. Cells were then cultured for a further 8 days in fresh medium. Finally, cells were fixed with 4% PFA in PBS, washed with PBS and incubated at 37 °C for 36 h with SA-β-galactosidase-staining solution (1 mg/mL 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside [X-gal], 5 mM K₃Fe[CN]₆, 5 mM K₄Fe[CN]₆, 2 mM MgCl₂ in PBS pH 6). Blue-green cell cytoplasmic staining, indicative of cell senescence due to an increase of lysosomal β-galactosidase activity, was examined under phase-contrast microscopy, and representative images were taken.

Cellular autofluorescence was determined by incubating ME180, MS751, and SCC152 cells (5 × 10⁴) for 120 h on 6-well plates in the presence of test compounds (50 μM). Cells were then harvested and changes in autofluorescence were assessed by flow cytometry detection in the FITC (green) channel using a LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The acquired data were analyzed by the Flowing software.

To evaluate apoptosis induction, ME180, MS751, and SCC152 cells were incubated on 6-well plates for 48, 72, 96, or 120 h (seeding 2 × 10⁵, 1 × 10⁵, 7.5 × 10⁴, or 5 × 10⁴, respectively) in the presence of each test compound. Cells were then harvested and AnnexinV-FITC binding along with PI staining (eBioscience™ Annexin V Apoptosis Detection Kit FITC, Thermo Fisher Scientific, Waltham, MA, USA) was analyzed by flow cytometry using a LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data analysis was carried out using the FACSDiva software (BD Biosciences, Franklin Lakes, NJ, USA).