High-content assay to measure mitochondrial function and bacterial vacuole size in infected human primary macrophages

Mariatou Dramé; Francisco Javier Garcia-Rodriguez; Carmen Buchrieser; Pedro Escoll

doi:10.1016/j.xpro.2023.102175

High-content assay to measure mitochondrial function and bacterial vacuole size in infected human primary macrophages

STAR Protoc. 2023 Mar 17;4(2):102175. doi: 10.1016/j.xpro.2023.102175. Online ahead of print.

Authors

Mariatou Dramé¹, Francisco Javier Garcia-Rodriguez¹, Carmen Buchrieser², Pedro Escoll³

Affiliations

¹ Institut Pasteur, Université Paris Cité, CNRS UMR 6047, Biologie des Bactéries Intracellulaires, 75015 Paris, France.
² Institut Pasteur, Université Paris Cité, CNRS UMR 6047, Biologie des Bactéries Intracellulaires, 75015 Paris, France. Electronic address: cbuch@pasteur.fr.
³ Institut Pasteur, Université Paris Cité, CNRS UMR 6047, Biologie des Bactéries Intracellulaires, 75015 Paris, France. Electronic address: pescoll@pasteur.fr.

Abstract

Regulation of bioenergetics and cell death are pivotal mitochondrial functions determining the responses of macrophages to infection. Here, we provide a protocol to investigate mitochondrial functions during infection of macrophages by intracellular bacteria. We describe steps for quantifying mitochondrial polarization, cell death, and bacterial infection in infected, living, human primary macrophages at the single-cell level. We also detail the use of the pathogen Legionella pneumophila as model. This protocol can be adapted to investigate mitochondrial functions in other settings. For complete details on the use and execution of this protocol, please refer to Escoll et al. (2021).¹.

Keywords: Cell Biology; Cell-based Assays; High-throughput Screening; Immunology; Metabolism; Microbiology; Microscopy; Single Cell.