Apoptosis Detection via Fluorescence Microscopy and Flow Cytometry

To detect apoptotic cell death, cells were pre-incubated with Hoechst 33342 (Invitrogen; Thermo Fisher Scientific, Inc.) for 30 min before treatment with compounds. Next, blue nuclei showing condensed or fragmented chromatin were analyzed using fluorescence microscopy (Leica Microsystems, Wetzlar, Germany) as reported [15 (link)]. The quantification of apoptotic cell death percentage was determined via flow cytometry analysis using an Allophycocyanin (APC) Annexin V conjugate and propidium iodide (Annexin V-APC/PI) staining. For these experiments, HCT116 cells (2 × 10⁵/2 mL medium) were seeded into 6-well plates and then subjected to treatments with MG. At the end of the treatment, the cells were taken via trypsinization, centrifuged at 120× g for 10 min, resuspended in PBS and counted. Next, 10⁵ cells were incubated with Annexin V-APC (BD Pharmingen™ APC Annexin V kit, BD Biosciences, Milan, Italy) and PI (Sigma-Aldrich) in the dark according to the manufacturers’ instructions. At the end of the incubation, the samples were analyzed using a FACSAria Cell Sorter flow cytometer (BD Biosciences Company, 283 Franklin Lakes, NJ, USA), acquiring at least 50,000 cells for each sample analyzed. The data obtained were then examined with FlowJo software (BD Biosciences).