Methods for Monitoring Mitochondrial Functions During Pathogen Infection

1st Edition, Volume 194 - March 10, 2025
Imprint: Academic Press
Editors: Saverio Marchi, Lorenzo Galluzzi
Language: English
Hardback ISBN:
9 7 8 - 0 - 3 2 3 - 9 9 2 1 9 - 0
eBook ISBN:
9 7 8 - 0 - 3 2 3 - 9 9 2 2 0 - 6

Methods for Monitoring Mitochondrial Functions During Pathogen Infection, Volume 194 in the Methods in Cell Biology series highlights advances in the field, with this new volume… Read more

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Methods for Monitoring Mitochondrial Functions During Pathogen Infection, Volume 194 in the Methods in Cell Biology series highlights advances in the field, with this new volume presenting interesting chapters on timely topics, including Mitochondrial Labeling with Fluorescent Proteins for Enhanced Morphological Characterization and Visualization of Intercellular Mitochondrial Transfer, Visualizing mitochondrial electron transport chain complexes and super-complexes during infection of human macrophages with Legionella pneumophila, Quantitative assessment of mitochondrial membrane potential in macrophages in sepsis, Quantification of mitochondrial reactive oxygen species in macrophages during sepsis, and much more.

Additional chapters cover Quantification of intracellular and mitochondrial ATP content in macrophages during lipopolysaccharide-induced inflammatory response, Analysis of cytosolic mtDNA release during Staphylococcus Aureus infection, Monitoring mitochondria dynamics upon infection using a tomographic approach, A screening system to determine bacterial effects on MADCAM1 expression by high endothelial venule cells, Isolation of mitochondria from Mycobacteria infected cells, Monitoring mitochondrial localization of Mycobacterial proteins, and more.

Methods for Monitoring Mitochondrial Functions During Pathogen Infection
Cover image
Title page
Table of Contents
Series Page
Copyright
Contributors
Chapter One Labeling of mitochondria for detection of intercellular mitochondrial transfer
Abstract
Keywords
1 Introduction
2 Materials
2.1 Common disposable
2.2 Cells and reagents
2.3 Equipment
3 Methods
3.1 Reagent preparations
3.2 Lentiviral production
3.3 Lentiviral transduction
3.4 Mitochondria transfer
3.5 Image acquisition
4 Notes
5 Concluding remarks
Appendix
References
Chapter Two Visualizing mitochondrial electron transport chain complexes and super-complexes during infection of human macrophages with Legionella pneumophila
Abstract
Keywords
1 Introduction
2 Materials
2.1 Disposables
2.2 Cells and reagents
2.3 Equipment
3 Methods
3.1 Cell culture, preparation of bacteria and infection
3.2 Fluorescence-activated cell (FACS) sorting of infected cells (see Note 5)
3.3 Isolation of mitochondria from sorted cells
3.4 Assessment of the integrity and purity of isolated mitochondria using a mitochondrial dye and flow cytometry
3.5 Visualization of mitochondrial ETC complexes and super-complexes by BN-PAGE and NIR Western blot (see Note 12)
3.6 Interpretation of the results
4 Notes
5 Concluding remarks
Conflicts of interest
References
Chapter Three Quantitative assessment of mitochondrial membrane potential in macrophages in sepsis
Abstract
Keywords
1 Introduction
2 Materials
2.1 Common disposables
2.2 Cells and reagents
2.3 Equipment
2.4 Software
3 Method
3.1 Isolation and culture of bone marrow-derived macrophages (BMDMs)
3.2 Culture of J774A.1 murine macrophage
3.3 RNA interference and treatment
3.4 Measurements of mitochondrial membrane potential
3.5 Data analysis
4 Notes
5 Concluding remarks
Competing interests
References
Chapter Four Quantification of mitochondrial reactive oxygen species in macrophages during sepsis
Abstract
Keywords
1 Introduction
2 Materials
2.1 Common disposables
2.2 Surgical instruments
2.3 Cells and reagents
2.4 Equipment
2.5 Software
3 Methods
3.1 Maintenance of mice
3.2 Culture of L929 cells
3.3 Isolation of bone marrow-derived macrophages from mice
3.4 Culture of bone marrow-derived macrophages
3.5 Small interfering RNA transfection
3.6 Preparation of imaging cell culture slide
3.7 Determination of mitochondrial ROS production
3.8 Data analysis
4 Notes
5 Concluding remarks
Competing interests
References
Chapter Five Quantification of intracellular and mitochondrial ATP content in macrophages during lipopolysaccharide-induced inflammatory response
Abstract
Keywords
1 Introduction
2 Materials
2.1 Common disposables
2.2 Surgical accessories
2.3 Cells and reagents
2.4 Equipment
2.5 Software
3 Methods
3.1 Preparation of L929 conditional medium
3.2 Extraction and culture of murine bone marrow-derived macrophages (BMDMs)
3.3 Measurement of intracellular ATP
3.4 Isolation of mitochondria from BMDM cells
3.5 Measurement of mitochondrial ATP
3.6 Data analysis
4 Notes
5 Concluding remarks
Competing interests
References
Chapter Six Analysis of cytosolic mtDNA release during Staphylococcus aureus infection
Abstract
Keywords
1 Introduction
2 Materials
2.1 Common disposable (see Notes 4.1 and 4.2)
2.2 Cells and reagents
2.3 Equipment
3 Methods
3.1 Reagent preparation
3.2 Cell culture and treatments
3.3 mtDNA extraction from whole cells (see Notes 4.2)
3.4 mtDNA extraction from the cytosol (see Note 4.14)
3.5 RT-PCR (see Notes 4.2 and 4.16)
3.6 Data analysis
4 Notes
5 Concluding remarks
Conflicts of interest
References
Chapter Seven Monitoring cellular dynamics upon infection using a holotomography-based approach
Abstract
Keywords
1 Introduction
2 Materials
2.1 Common disposables (see Note 4.1)
2.2 Cells and reagents (see Note 4.1)
2.3 Equipment
3 Methods
3.1 Bacterial growth and inoculum preparation
3.2 Cell analysis upon infection using holotomographic approach
4 Notes
5 Concluding remarks
Conflicts of interest
References
Chapter Eight A screening system to determine the effect of bacterial metabolites on MAdCAM-1 expression by transformed endothelial sinusoidal cells
Abstract
Keywords
1 Introduction
2 Materials and methods
2.1 Cell lines and culture reagents
2.2 Cell culture procedures
2.3 Sub-culture procedures
2.4 Establishment of Geneticin™ selection condition
2.5 MAdCAM-1 promoter plasmid
2.6 MAdCAM-1 promoter reporter cell line
2.7 Image acquisition and processing
2.8 Data analysis
3 Concluding remarks
4 Notes
Disclosures
References
Chapter Nine Monitoring the mitochondrial localization of mycobacterial proteins
Abstract
Keywords
1 Introduction
2 Materials
2.1 Disposables
2.2 Cells and reagents
2.3 Equipment
2.4 Software
3 Methods
3.1 Expression of a candidate gene in mycobacterial vector
3.2 Expression of the candidate gene in mammalian vector
3.3 Transformation of Mycolicibacterium smegmatis
3.4 Culture of host cells
3.5 Mycobacterial culture and infection
3.6 Transfection
3.7 Imaging of mycobacterial infection
3.8 Fluorescence staining and imaging
3.9 Isolation of mitochondria
3.10 Immunoblotting analyses
4 Concluding remarks
References
Chapter Ten Bacterial predators and BALOs: Growth protocol and relation with mitochondria
Abstract
Keywords
1 Introduction
2 Materials
2.1 Common disposables
2.2 Cells and reagents
2.3 Equipment (see Note 1)
2.4 Softwares (see Note 1)
3 Methods
3.1 Growth and propagation of bacterial strains
3.2 Preparation of B. bacteriovorus suspension for predatory assays (see Note 7)
3.3 Predation assays on prey cultures
3.4 Predatory activity of B. bacteriovorus on static biofilms
3.5 PCR procedure work-flow
4 Notes
5 Concluding remarks
References
Chapter Eleven Assessment of adhering and invading properties of Escherichia coli strains
Abstract
Keywords
1 Introduction
2 Materials
2.1 Common disposables
2.2 Cells and reagents
2.3 Equipment (see Note 1)
3 Methods
3.1 E. coli strains isolation from intestinal biopsies (see Note 3)
3.2 Adhesion assay
3.3 Invasion assay
4 Notes
5 Concluding remarks
References

Saverio Marchi

Saverio Marchi obtained his PhD in ‘Pharmacology and Molecular Oncology’ from University of Ferrara (Italy), under the supervision of Prof. Rosario Rizzuto, where he investigated the role of Ca2+ signalling and mitochondrial dynamics in cancer. He did a short term research stay at the Gustave Roussy Institute (Paris, France) in the Guido Kroemer’s lab, for the study of the mechanisms of autophagy regulation. On 2011 he joined the Laboratory of Dr. Paolo Pinton at the University of Ferrara (Italy) where he carried out his postdoctoral studies. During his postdoctoral training, he studied the implications of Endoplasmic Reticulum-mitochondria connection in diseases, especially cancer. In 2014, he was the recipient of the Young Researcher award, which allowed him to work at the Cancer Center, University of Hawai’i (Prof. Michele Carbone’s lab), where he was introduced to the study of the signalling pathways regulating mesothelioma progression. Now, he is associate professor at the Marche Polytechnic University, where he leads his own group. He is interested in the involvement of the MCU complex and mitochondrial Ca2+ in the evolution of cancer. More recently, he has become interested in the alteration of mitochondrial dynamics during bacterial infection. He published 77 papers on “peer-reviewed” journals (h-index: 41; n° of citations > 5800. Source: Scopus).

Affiliations and expertise

Dept. of Clinical and Molecular Sciences, Marche Polytechnic University, Ancona, Italy

Lorenzo Galluzzi

Lorenzo Galluzzi is Assistant Professor of Cell Biology in Radiation Oncology at the Department of Radiation Oncology of the Weill Cornell Medical College, Honorary Assistant Professor Adjunct with the Department of Dermatology of the Yale School of Medicine, Honorary Associate Professor with the Faculty of Medicine of the University of Paris, and Faculty Member with the Graduate School of Biomedical Sciences and Biotechnology of the University of Ferrara, the Graduate School of Pharmacological Sciences of the University of Padova, and the Graduate School of Network Oncology and Precision Medicine of the University of Rome “La Sapienza”. Moreover, he is Associate Director of the European Academy for Tumor Immunology and Founding Member of the European Research Institute for Integrated Cellular Pathology. Galluzzi is best known for major experimental and conceptual contributions to the fields of cell death, autophagy, tumor metabolism and tumor immunology. He has published over 450 articles in international peer-reviewed journals and is the Editor-in-Chief of four journals: OncoImmunology (which he co-founded in 2011), International Review of Cell and Molecular Biology, Methods in Cell biology, and Molecular and Cellular Oncology (which he co-founded in 2013). Additionally, he serves as Founding Editor for Microbial Cell and Cell Stress, and Associate Editor for Cell Death and Disease, Pharmacological Research and iScience.

Affiliations and expertise

Assistant Professor of Cell Biology in Radiation Oncology, Department of Radiation Oncology, Weill Cornell Medical College, NY, USA

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