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.

Autor:	Dhyani V; Bioimaging and Data Analysis Lab, Department of Chemical Engineering, Indian Institute of Technology Hyderabad, Sangareddy, Telangana, India.; Optical Science Centre, Faculty of Science, Engineering & Technology, Swinburne University of Technology, Hawthorn, Australia., Chann AS; Optical Science Centre, Faculty of Science, Engineering & Technology, Swinburne University of Technology, Hawthorn, Australia.; Immune Signalling Laboratory, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia.; Department of Immunology, Mayo Clinic, Scottsdale, AZ, USA., Giri L; Bioimaging and Data Analysis Lab, Department of Chemical Engineering, Indian Institute of Technology Hyderabad, Sangareddy, Telangana, India., Russell SM; Optical Science Centre, Faculty of Science, Engineering & Technology, Swinburne University of Technology, Hawthorn, Australia. sarah.russell@petermac.org.; Immune Signalling Laboratory, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia. sarah.russell@petermac.org.; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, VIC, Australia. sarah.russell@petermac.org., Charnley M; Optical Science Centre, Faculty of Science, Engineering & Technology, Swinburne University of Technology, Hawthorn, Australia. mcharnley@swin.edu.au.; Immune Signalling Laboratory, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia. mcharnley@swin.edu.au.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2024; Vol. 2800, pp. 167-187.
DOI:	10.1007/978-1-0716-3834-7_12
Abstrakt:	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. (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze:	MEDLINE
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