Apoptosis Assays for Cancer Cell Lines

Cell apoptosis was determined using the terminal deoxynucleotidyl transferase (TdT) deoxyuridine dUTP nick-end labeling (TUNEL) and Annexin V assays as previously described (Kee et al., 2017 (link)). CT26 and HT29 cells were seeded in six-well plates, treated with G.A for 24 h, fixed with 3.7% paraformaldehyde for 20 min, and then washed with phosphate-buffered saline (PBS). The cells were incubated with 4′,6-diamidino-2-phenylindole (DAPI) solution (2.5 μg/mL) for 3 min at room temperature and washed with PBS. Apoptotic cells and stained nuclei were observed using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). In addition, the apoptotic cells were analyzed using the MUSE Annexin V and dead cell kit (Millipore, Billerica, MA, United States) in accordance with the recommended protocol. The stained cells were analyzed using the Muse cell analyzer.