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
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