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Quantifying Regulated Mitochondrial Fission in Macrophages

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Effector-Triggered Immunity

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2523))

Abstract

Mitochondria have co-evolved with eukaryotic cells for more than a billion years, becoming an important cog in their machinery. They are best known for being tasked with energy generation through the production of adenosine triphosphate, but they also have roles in several other cellular processes, for example, immune and inflammatory responses. Mitochondria have important functions in macrophages, key innate immune cells that detect pathogens and drive inflammation. Mitochondrial activity is influenced by the highly dynamic nature of the mitochondrial network, which alternates between interconnected tubular and fragmented forms. The dynamic balance between this interconnected fused network and fission-mediated mitochondrial fragmentation modulates inflammatory responses such as production of cytokines and mitochondrial reactive oxygen species. Here we describe methods to differentiate mouse bone marrow cells into macrophages and the use of light microscopy, electron microscopy, flow cytometry, and Western blotting to quantify regulated mitochondrial dynamics in these differentiated macrophages.

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Acknowledgments

RK received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 894690, a Rebecca L. Cooper grant (#021765). MJS is an NHMRC Leadership Fellow, supported by an NHMRC Investigator Grant (APP1194406) and an NHMRC project grant (APP1125316). SFA is supported by an Australian Government Research Training Program (RTP) Scholarship. NDC is supported as a CZI Imaging Scientist by grant number 2020-225648 from the Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation. Microscopy was performed at the Australian Cancer Research Foundation (ACRF)/Institute for Molecular Bioscience Cancer Biology Imaging. We thank Professor Robert Parton for insightful discussions around best practice for electron microscopy imaging and analysis.

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Authors and Affiliations

  1. Institute for Molecular Bioscience (IMB), The University of Queensland, Brisbane, QLD, Australia

    Syeda Farhana Afroz, Nicholas D. Condon, Matthew J. Sweet & Ronan Kapetanovic

  2. IMB Centre for Inflammation and Disease Research, The University of Queensland, Brisbane, QLD, Australia

    Syeda Farhana Afroz, Matthew J. Sweet & Ronan Kapetanovic

  3. Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, QLD, Australia

    Syeda Farhana Afroz, Matthew J. Sweet & Ronan Kapetanovic

  4. Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland

    Ronan Kapetanovic

Authors
  1. Syeda Farhana Afroz
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  2. Nicholas D. Condon
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  3. Matthew J. Sweet
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  4. Ronan Kapetanovic
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Corresponding authors

Correspondence to Matthew J. Sweet or Ronan Kapetanovic .

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Editors and Affiliations

  1. Department of Immunology, Institute of Nutritional Medicine, University of Hohenheim, Stuttgart, Baden-Württemberg, Germany

    Thomas A. Kufer

  2. Department of Physiology Anatomy & Microbiology, La Trobe University, Bundoora, VIC, Australia

    Maria Kaparakis-Liaskos

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Afroz, S.F., Condon, N.D., Sweet, M.J., Kapetanovic, R. (2022). Quantifying Regulated Mitochondrial Fission in Macrophages. In: Kufer, T.A., Kaparakis-Liaskos, M. (eds) Effector-Triggered Immunity. Methods in Molecular Biology, vol 2523. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2449-4_18

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  • DOI: https://doi.org/10.1007/978-1-0716-2449-4_18

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2448-7

  • Online ISBN: 978-1-0716-2449-4

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