Link to Pubmed [PMID] – 40058960
Link to DOI – 10.1016/bs.mcb.2024.01.003
Methods Cell Biol 2025 ; 194(): 19-42
The ultrastructure of mitochondria is pivotal for their respiratory activity. Thus, the regulation of the assembly of the super-complexes (SCs) of the mitochondrial electron transport chain (ETC) might be a core aspect of macrophage immunometabolism during bacterial infection. In order to study the impact of infection by Legionella pneumophila on the configuration of mitochondrial complexes and SCs in human macrophages, we have adapted and combined different methods such as cell sorting of infected cells, magnetic isolation of highly pure and functional mitochondria, quality control of mitochondrial purity by flow cytometry, and BN-PAGE (Blue-Native Polyacrylamide Gel Electrophoresis) coupled to Western Blot using near-infrared (NIR) fluorescence. The here presented protocol uses infected and non-infected human macrophage-like THP-1 cells and GFP-expressing L. pneumophila, but the method can be used to analyze the configuration of ETC complexes and SCs also in other mammalian cells and infected with different intracellular bacteria expressing a fluorescent protein.