2.3. Cell Apoptosis Analysis by Flow Cytometry and DAPI Staining Assay

Cells cultured in 6-well plates were treated with phloretin (0, 20, 50, and 100 μM) for 24 h and then harvested for flow cytometry analysis (cell apoptosis assay) by using an Annexin V-FITC Apoptosis Detection Kit (Keygentec, Nanjing, China) according to the instruction of manufacturer. Flow cytometry analysis for cell apoptosis was performed using the EPICS Elite ESP high-performance cell sorter (Coulter Electronics, Ltd., England, UK) and analyzed by ModFit LT (version 2.0; Verity Software), and a minimum of 30,000 events were collected for each sample.

For DAPI staining assay, cells cultured in 12-well plates were incubated with phloretin (0, 20, 50, and 100 μM) for 24 h. Cells were briefly washed with 1× PBS and fixed in 4% formaldehyde for 15 min, and then washed three times with 1× PBS and permeabilized in 0.2% Triton X-100 for 15 min. Finally, cells were stained with DAPI (1 μg/ml) at 37°C for 30 min in the dark and then observed and photographed by fluorescence microscopy (Nikon, IX-71, Japan).