A Pipeline for Dynamic Analysis of Mitochondrial Content in Developing T Cells: Bridging the Gap Between High-Throughput Flow Cytometry and Single-Cell Microscopy Analysis
Journal Title
In: Wuelfing, C., Murphy, R.F. (eds) Imaging Cell Signaling. Methods in Molecular Biology, vol 2800
Publication Type
Protocol
Abstract
Analyzing the dynamics of mitochondrial content in developing T cells is crucial for understanding the metabolic state during T cell development. However, monitoring mitochondrial content in real-time needs a balance of cell viability and image resolution. In this chapter, we present experimental protocols for measuring mitochondrial content in developing T cells using three modalities: bulk analysis via flow cytometry, volumetric imaging in laser scanning confocal microscopy, and dynamic live-cell monitoring in spinning disc confocal microscopy. Next, we provide an image segmentation and centroid tracking-based analysis pipeline for automated quantification of a large number of microscopy images. These protocols together offer comprehensive approaches to investigate mitochondrial dynamics in developing T cells, enabling a deeper understanding of their metabolic processes.
Keywords
*Flow Cytometry/methods; *Mitochondria/metabolism; *Single-Cell Analysis/methods; *T-Lymphocytes/metabolism/cytology; *Microscopy, Confocal/methods; Animals; Image Processing, Computer-Assisted/methods; Humans; Mice; Mitochondrial Dynamics; 3D imaging; Dn3; Density-based spatial clustering of applications with noise (DBSCAN); Image segmentation; MitoView Green; Mitochondria
Department(s)
Laboratory Research
PubMed ID
38709484
Terms of Use/Rights Notice
Refer to copyright notice on published article.


Creation Date: 2024-08-20 03:42:06
Last Modified: 2024-08-20 06:57:07

© 2024 The Walter and Eliza Hall Institute of Medical Research. Access to this website is subject to our Privacy Policy and Terms of Use

An error has occurred. This application may no longer respond until reloaded. Reload 🗙