Single cell temporally-resolved analysis of apoptotic and non-apoptotic cell death by live-cell imaging

Apoptosis and other regulated cell death pathways display complex, dynamic, and heterogeneous behaviors that are frequently masked by conventional endpoint assays relying on bulk measurements. Here, we describe a live-cell imaging workflow for temporally resolved, single-cell quantification of both apoptotic and non-apoptotic cell death in cell cultures. In colorectal carcinoma cell lines, cell death was triggered by agents including Staurosporine, RSL3, and Cisplatin, and continuously tracked using a live-cell imaging platform with fluorescent markers such as Annexin V-FITC, Caspase-3/7 activity dye, and propidium iodide. Parameters of cell death were monitored for 24-48 h, allowing precise discrimination between early and late apoptotic events as well as necrosis. This methodology enables reproducible, quantitative profiling of cell death kinetics at single-cell resolution, reduces artifacts from manual handling, and offers valuable insights into the temporal dynamics and interplay of different cell death modalities.