活细胞成像技术可用于可视化调控细胞死亡过程中 DAMPs 的释放。

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

机构信息

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo City, Tokyo, Japan.

出版信息

Methods Mol Biol. 2021;2274:337-352. doi: 10.1007/978-1-0716-1258-3_28.

Abstract

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.

摘要

本方案介绍了一种活细胞分泌活动成像（LCI-S）技术，该技术使用免疫测定法与全内反射荧光（FL）显微镜结合，可用于实时观察单个细胞中分子的释放。这种新的“活”细胞成像技术通过使用这种新方法帮助揭示了受调控的细胞“死亡”的动力学。通过针对表达荧光共振能量转移（FRET）生物传感器的细胞中外源释放损伤相关分子模式（DAMPs），该方案可以通过单细胞成像观察受调控的细胞死亡过程的最后阶段，例如基于 FRET 的 RIPK3 激活的 MLKL 传感器（SMART）和基于 FRET 的 caspase-1 激活传感器（SCAT1），它们可特异性识别受调控的细胞死亡过程的发生。