Microscope system ix51

The production of intracellular ROS was measured using the redox-sensitive fluorescent dye 2′-7′-dichlorofluorescein diacetate [DCF-DA (C₂₄H₁₄C_l2O₇); Sigma-Aldrich, Inc., St. Louis, MO, USA]. The ability of cells to produce ROS was measured by fluorescence. The cells were treated with 25 μM DCF-DA for 30 min at 37°C and washed twice in PBS. Representative images were obtained using a fluorescence microscope (Olympus Microscope System IX51; Olympus).

matrigel invasion assays were used to assess the effect of glaucine in MCF-7 or MDA-MB-231 cells. The 8-µm pore-size polycarbonate nucleopore filter inserts in a 24-well transwell chamber (BD Biosciences, San Jose, CA, USA) was coated with 30 µg/well matrigel (Sigma). Glaucine-treated MCF-7 cells were seeded into the upper part of the matrigel-coated filter, and serum-free RPMI with or without 100-nM PMA was added to the lower part. After 36 h, the cells that had migrated through the matrigel and the 8-µm pore-size membrane were fixed, stained, and counted under a light microscope (Olympus Microscope System IX51; Olympus, Tokyo, Japan).

SA-β-gal activity was determined at 24 h after UVA irradiation. A cellular senescence assay kit (Cell Biolabs, Inc., San Diego, CA, USA) was performed according to the manufacturer’s instructions. Briefly, the cells were washed twice in PBS and incubated at room temperature for 5 min with fixing solution. The cells were washed three times with PBS, the final wash was aspirated, and the cells were completely covered with freshly prepared cell staining working solution. The cells were then incubated in the dark overnight at 37°C. Following removal of the cell staining solution, the cells were washed twice with PBS and blue-stained senescence cells were observed using a light microscope (Olympus Microscope System IX51; Olympus, Tokyo, Japan).