Live-Cell Imaging Technique to Visualize DAMPs Release During Regulated Cell Death.

The present protocol introduces a live-cell imaging of secretion activity (LCI-S) that is useful to visualize the real-time release of molecules from individual cells using an immunoassay coupled with total internal reflection fluorescence (FL) microscopy. This novel "live"-cell imaging technique has helped uncover the dynamics of regulated cell "death" by using this new approach. This protocol can observe the final stages of the regulated cell death process via single-cell imaging by targeting the extracellular release of damage-associated molecular patterns (DAMPs) from the cells expressing fluorescence resonance energy transfer (FRET) biosensors, such as a sensor for MLKL activation by RIPK3 based on FRET (SMART) and a sensor for caspase-1 activation based on FRET (SCAT1), which specifically identify the occurrence of regulated cell death processes.