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

Methods Mol Biol. 2021;2274:353-363. doi: 10.1007/978-1-0716-1258-3_29.


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.

Keywords: DAMPs; FRET; HMGB1; LCI-S; Necroptosis; SMART.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alarmins / metabolism*
  • Fibroblasts / metabolism
  • Fibroblasts / pathology*
  • Fluorescence Resonance Energy Transfer / methods*
  • HMGB1 Protein / metabolism*
  • Humans
  • Necroptosis*
  • Protein Kinases / metabolism*
  • Time-Lapse Imaging / methods*


  • Alarmins
  • HMGB1 Protein
  • HMGB1 protein, human
  • MLKL protein, human
  • Protein Kinases