使用 mt-Keima 监测线粒体自噬的方法。

Methods for Monitoring Mitophagy Using mt-Keima.

机构信息

Inherited Movement Disorders Unit, Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, Division of Intramural Research, National Institutes of Health, Bethesda, MD, USA.

出版信息

Methods Mol Biol. 2024;2845:151-160. doi: 10.1007/978-1-0716-4067-8_12.

DOI:10.1007/978-1-0716-4067-8_12

PMID:39115664

Abstract

Mitochondria-targeted Keima (mt-Keima) is a pH-sensitive, acid-stable fluorescent protein used for the quantification of mitophagy. Mt-Keima contains a mitochondrial matrix targeting sequence and has bimodal excitation with peaks at 440 nM in neutral environments and 586 nM in acidic environments. From this bimodal excitation, a ratiometric signal may be calculated to quantify mitophagy in live cells. This chapter describes procedures for measuring mitophagy by flow cytometry and live cell confocal microscopy with mt-Keima.

摘要

线粒体靶向 Keima（mt-Keima）是一种 pH 敏感、耐酸的荧光蛋白，用于定量监测自噬体。mt-Keima 含有一个线粒体基质靶向序列，在中性环境中具有双峰激发峰，峰值在 440nm，在酸性环境中激发峰在 586nm。通过这种双峰激发，可以计算出比率信号来定量活细胞中的自噬体。本章描述了使用 mt-Keima 通过流式细胞术和活细胞共聚焦显微镜测量自噬体的步骤。