Tobias I C, Khazaee R, Betts D H
Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, Western University, London, Ontario, Canada.
Lawson Health Research Institute, Western University, London, Ontario, Canada.
Curr Protoc Stem Cell Biol. 2018 Nov;47(1):e67. doi: 10.1002/cpsc.67. Epub 2018 Oct 10.
Dynamic alterations to mitochondrial structure and function regulate cell fate decisions and underlie multiple age-related and genetic diseases that are modeled using embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Transmission electron microscopy (TEM) can be used to obtain high-resolution micrographs of mitochondria, but mitochondrial ultrastructure is easily distorted during specimen processing. This unit describes a method that preserves mitochondrial membrane structure from adherent ESC cultures for TEM sample preparation. This procedure is useful for assessing ultrastructural changes to mitochondria during differentiation, reprogramming, or experimental manipulation of ESC metabolism. We provide comprehensive protocols for: (1) preparation of ESC cultures for TEM; (2) retrieval of thin sections from individual ESCs; and (3) contrast staining and morphometric analysis of mitochondria and cristae. This unit also describes an alternative procedure for samples with low cell numbers and a supporting protocol for morphometric image analysis. Collectively, these protocols allow for the observation and quantitative analysis of mitochondria in ESCs. © 2018 by John Wiley & Sons, Inc.
线粒体结构和功能的动态变化调节细胞命运决定，并构成多种与年龄相关和遗传疾病的基础，这些疾病可通过胚胎干细胞（ESC）和诱导多能干细胞（iPSC）进行建模。透射电子显微镜（TEM）可用于获取线粒体的高分辨率显微照片，但线粒体超微结构在标本处理过程中容易变形。本单元介绍了一种用于TEM样品制备的方法，该方法可保留贴壁ESC培养物中的线粒体膜结构。此程序对于评估ESC分化、重编程或代谢实验操作过程中线粒体的超微结构变化很有用。我们提供了全面的方案，包括：（1）用于TEM的ESC培养物制备；（2）从单个ESC中获取薄片；（3）线粒体和嵴的对比染色及形态计量分析。本单元还描述了细胞数量少的样品的替代程序以及形态计量图像分析的支持方案。总体而言，这些方案可用于观察和定量分析ESC中的线粒体。© 2018约翰威立国际出版公司
