Live-Cell Imaging Technique to Visualize DAMPs Release During Regulated Cell Death
Mai Yamagishi1, Yoshitaka Shirasaki2
1Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo City, Tokyo, Japan.
Methods in Molecular Biology (Clifton, N.J.)
|May 29, 2021
Summary
This study presents live-cell imaging of secretion activity (LCI-S) to visualize real-time molecular release and uncover regulated cell death dynamics using fluorescence microscopy and biosensors.
Area of Science:
- Cell biology
- Molecular imaging
- Immunology
Background:
- Regulated cell death (RCD) involves complex molecular events.
- Understanding RCD dynamics requires advanced imaging techniques.
- Extracellular release of damage-associated molecular patterns (DAMPs) is a hallmark of RCD.
Purpose of the Study:
- To introduce a novel live-cell imaging of secretion activity (LCI-S) protocol.
- To visualize real-time molecular release from individual cells during RCD.
- To investigate the dynamics of RCD using single-cell imaging.
Main Methods:
- Coupling immunoassay with total internal reflection fluorescence (TIRF) microscopy for LCI-S.
- Utilizing fluorescence resonance energy transfer (FRET) biosensors to detect specific RCD events.
- Employing biosensors such as SMART (for MLKL activation) and SCAT1 (for caspase-1 activation).
Main Results:
- LCI-S successfully visualizes real-time molecular release during RCD.
- The technique allows for single-cell imaging of RCD final stages.
- Demonstrated visualization of DAMPs release using FRET biosensors.
Conclusions:
- LCI-S is a powerful tool for studying RCD dynamics at the single-cell level.
- This method provides novel insights into the molecular mechanisms of regulated cell death.
- The protocol facilitates the study of specific RCD pathways through targeted biosensor imaging.
