Apoptosis analysis via Hoechst staining

The MCF-7 and HepG2 cells were seeded into 6-well plates at a density of 2×10⁵ cells/well. The cells were treated with CM at 0.5, 1.0 and 2.0 mg/ml for 24 h. The treated cells were then incubated with Hoechst 33342 (5 µg/ml; BD Biosciences, San Jose, CA, USA) for 30 min at 37°C in the dark. Following three washes with phosphate-buffered saline (PBS), images of the altered fluorescent color in the mitochondria were captured using a fluorescent microscope (magnification, x20; charge-coupled device camera; TE2000; Nikon Corporation, Tokyo, Japan). The percentage of apoptotic cells was analyzed by measuring the fluorescence intensity using Image J software (rsb.info.nih.gov/ij/download.html) and expressed as the ratio of red to green fluorescence intensity.