Apoptosis inhibition provides survival and proliferative advantages to tumor cells. Therefore induction of apoptosis is a critical mode of action for several anti-tumor and chemotherapy agents. While several well-established methods for evaluating apoptosis are available, most offer single, user-defined endpoint readouts and are not amenable to long-term measurements in live cells that can provide mechanistic insights into the biochemical and morphological changes associated with the process.
A recent paper in Scientific Reports describes a protocol for real-time, live cancer cell imaging to assess three crucial mechanisms of apoptosis simultaneously, following drug treatment. Triple fluorescence staining with NucView®488 Caspase-3 Substrate, CF®594 AnnexinV, and MitoView™ Blue was used to monitor caspase-3 activity, phosphatidylserine exposure on the cell surface, and mitochondrial potential respectively. Colocalization studies show a characteristic staining pattern, where staining with MitoView™ Blue is mutually exclusive to both staining with NucView® 488 and CF®594 Annexin V, demonstrating that the protocol can accurately identify healthy (non-apoptotic) from apoptotic cells. Extensive validation of the protocol was performed using complementary methods including flow cytometry, western blotting and fluorescence imaging with other apoptotic and viability markers. This study has important implications in cancer research to decipher underlying apoptotic mechanisms in tumor cells after drug treatment.