Time-Lapse Imaging of Necroptosis and DAMP Release at Single-Cell Resolution.

Necroptosis is a regulated form of necrosis that depends on receptor-interacting protein kinase (RIPK)3 and mixed lineage kinase domain-like protein (MLKL). Necroptotic cells release a variety of cellular and nuclear factors, referred to as danger-associated molecular patterns (DAMPs). We recently developed a förster resonance energy transfer (FRET) biosensor, termed SMART (a sensor for MLKL activation based on FRET). SMART comprises a fragment of MLKL, and it monitors necroptosis, but not apoptosis or necrosis. We performed live-cell imaging for secretion activity (LCI-S) to observe the release of high-mobility group box 1 (HMGB1) from necroptotic cells at single-cell resolution. Moreover, we combined SMART and LCI-S imaging techniques and found two different modes of HMGB1 release from necroptotic cells. Thus, SMART and LCI-S are valuable tools for investigating intimate cross talk between necroptosis and DAMP release at single-cell resolution.