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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

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Imaging Cell Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2800))

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.

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Acknowledgments

This work was done on Wurundjeri land of the Kulin nation, and we pay our respects to the Elders past and present. Authors would like to acknowledge BioRender for their graphic design software.

Supporting Information

The supporting information, including additional data, code, and figures, is available on GitHub at https://github.com/VaibhavDhyani/Sample_codes_for_image_segmentation. Please refer to this link for further details.

Funding

National Health and Medical Research Council grant APP1099140 (SMR).

Flow cytometry was supported by a grant for the FACS Aria from the L.E.W Carty Charitable Fund.

CASS Foundation (CASS medicine/science grant number: 7818, MC).

Author information

Authors and Affiliations

  1. Bioimaging and Data Analysis Lab, Department of Chemical Engineering, Indian Institute of Technology Hyderabad, Sangareddy, Telangana, India

    Vaibhav Dhyani & Lopamudra Giri

  2. Optical Science Centre, Faculty of Science, Engineering & Technology, Swinburne University of Technology, Hawthorn, Australia

    Vaibhav Dhyani, Anchi S. Chann, Sarah M. Russell & Mirren Charnley

  3. Immune Signalling Laboratory, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia

    Anchi S. Chann, Sarah M. Russell & Mirren Charnley

  4. Department of Immunology, Mayo Clinic, Scottsdale, AZ, USA

    Anchi S. Chann

  5. Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, VIC, Australia

    Sarah M. Russell

Authors
  1. Vaibhav Dhyani
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  2. Anchi S. Chann
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  3. Lopamudra Giri
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  4. Sarah M. Russell
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  5. Mirren Charnley
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Corresponding authors

Correspondence to Sarah M. Russell or Mirren Charnley .

Editor information

Editors and Affiliations

  1. School of Cellular and Molecular Medicine, University of Bristol, Bristol, UK

    Christoph Wuelfing

  2. Computational Biology Department, Carnegie Mellon University, Pittsburgh, PA, USA

    Robert F. Murphy

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© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Dhyani, V., Chann, A.S., Giri, L., Russell, S.M., Charnley, M. (2024). 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. In: Wuelfing, C., Murphy, R.F. (eds) Imaging Cell Signaling. Methods in Molecular Biology, vol 2800. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3834-7_12

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  • DOI: https://doi.org/10.1007/978-1-0716-3834-7_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3833-0

  • Online ISBN: 978-1-0716-3834-7

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