Protocol for evaluating mitochondrial morphology changes in response to CCCP-induced stress through open-source image processing software

• Published in: STAR protocols 2023-12, Vol.4 (4), p.102745-102745, Article 102745
• Main Authors: Nag, Sudeshna, Szederkenyi, Kaitlin, Yip, Christopher M., McQuibban, G. Angus
• Format: Article
• Language: English
• Subjects: Carbonyl Cyanide m-Chlorophenyl Hydrazone - pharmacology; Cell Biology; Cell culture; Metabolism; Microscopy; Mitochondria - physiology; Molecular/Chemical Probes; Protocol; Software
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2023.102745

Mitochondrial morphology is an indicator of cellular health and function; however, its quantification and categorization into different subclasses is a complicated process. Here, we present a protocol for mitochondrial morphology quantification in the presence and absence of carbonyl cyanide m-chlorophenyl hydrazone stress. We describe steps for the preparation of cells for immunofluorescence microscopy, staining, and morphology quantification. The quantification protocol generates an aspect ratio that helps to categorize mitochondria into two clear subclasses. For complete details on the use and execution of this protocol, please refer to Nag et al.1 [Display omitted] •A quantification pipeline to study mitochondrial morphology•An unbiased pipeline has been created by using Fiji open-source image processing software•Applicable for characterizing mitochondrial morphology subclasses from confocal images Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Mitochondrial morphology is an indicator of cellular health and function; however, its quantification and categorization into different subclasses is a complicated process. Here, we present a protocol for mitochondrial morphology quantification in the presence and absence of carbonyl cyanide m-chlorophenyl hydrazone stress. We describe steps for the preparation of cells for immunofluorescence microscopy, staining, and morphology quantification. The quantification protocol generates an aspect ratio that helps to categorize mitochondria into two clear subclasses.