, Dimethylsulfoxide (DMSO): store at room temperature in the dark

, BSA

, PBS containing 1% FBS: add 5 mL of FBS in a 500 mL PBS bottle. Store at 4°C for up to one month

, 1% BSA (low endotoxin): add 0.5 mg of BSA in a 500 mL PBS bottle. Store at 4°C for up to one month, PBS containing 0

, Ficoll-Paque solution. Store at room temperature in the dark

, Anti-human CD14 antibodies coupled to magnetic beads (Miltenyi Biotec, cat# 130-050-201). Store at 4°C

, LS columns

Q. Separator,

. Macs-multistand,

, Nunc UpCell Surface cell culture, vol.6

, Recombinant human macrophage colony-stimulating factor: resuspend 25 µg of lyophilized rhMCSF in 500 µL of PBS + 0.1% BSA to have stock of 50 µg/mL. Make aliquots of 20 µL. Store at -20°C. Avoid freeze/thaw cycles

, Weight 29.2 mg of EDTA and dissolve it in 45 mL of PBS. Filter-sterilize by using a 0.22 µm PVDF membrane. Add 5 mL of FBS and sonicate for 30 min, PBMC-buffer: 2 mM EDTA + 10% FBS

, Complete RPMI medium: add 50 mL of FBS to a 500 mL bottle of RPMI 1640+Glutamax medium. Store at 4°C for up to one month

, Formulate the percentage of cells with fragmented mitochondria

, Mitochondrial activity: analysis of mitochondrial respiration (Seahorse) 1. The day of the assay

, Prepare the Assay Medium by supplementing Seahorse XF Base Medium with 1 mM pyruvate and 10 mM glucose

, Allow the compounds of the Seahorse XF Cell Mito Stress Test Kit to warm to room temperature in the sealed bag for approximately 15 minutes and prepare the compounds of the Seahorse XF Cell Mito Stress Test Kit as explained in section 2

, Load compounds in the ports of the Seahorse XFe96 Sensor Cartridges (from 3.2.19) using a 10-100 µL multichannel pipette and the corresponding loading guides as follows: Port A) Add 20 µL of 10X Olygomycin

, Port B) Add 22 µL of 10X FCCP

C. Port, Add 25 µL of 10X Rotenone+AntimycinA (from 3.8.6); and Port D) Add 28 µL of Assay Medium (see Note 11)

, Leave the Sensor Cartridge in the 37°C non-CO 2 incubator until use

, Design the assay file by specifying the Blank wells (no hMDMs, use the borders of the plate for these controls), the non-infected hMDMs samples and the Legionellainfected samples (all recommended by quadruplicate), as well as the routine for port injections and Mix-Wait-Measure cycles

A. P. West, G. S. Shadel, and S. Ghosh, Mitochondria in innate immune responses, Nat Rev Immunol, vol.11, pp.389-402, 2011.

P. Escoll, S. Mondino, M. Rolando, and C. Buchrieser, Targeting of host organelles by pathogenic bacteria: a sophisticated subversion strategy, Nat Rev Micro, vol.14, pp.5-19, 2016.
URL : https://hal.archives-ouvertes.fr/pasteur-01326394

P. Escoll, O. Song, and F. Viana, Legionella pneumophila Modulates Mitochondrial Dynamics to Trigger Metabolic Repurposing of Infected Macrophages, Cell Host Microbe, vol.22, pp.302-316, 2017.
URL : https://hal.archives-ouvertes.fr/pasteur-01687682

S. Banga, P. Gao, and X. Shen, Legionella pneumophila inhibits macrophage apoptosis by targeting pro-death members of the Bcl2 protein family, Proc Natl Acad Sci, vol.104, pp.5121-5126, 2007.

M. A. Wynosky-dolfi, A. G. Snyder, and N. H. Philip, Oxidative metabolism enables Salmonella evasion of the NLRP3 inflammasome, Journal of Experimental Medicine, vol.211, pp.653-668, 2014.

J. R. Friedman and J. Nunnari, Mitochondrial form and function, Nature, vol.505, pp.335-343, 2014.

K. Labbé, A. Murley, and J. Nunnari, Determinants and functions of mitochondrial behavior, Annu Rev Cell Dev Biol, vol.30, pp.357-391, 2014.

T. Wai and T. Langer, Mitochondrial Dynamics and Metabolic Regulation, Trends Endocrinol Metab, vol.27, pp.105-117, 2016.
URL : https://hal.archives-ouvertes.fr/hal-02391015

B. Westermann, Bioenergetic role of mitochondrial fusion and fission, Biochim Biophys Acta, vol.1817, pp.1833-1838, 2012.

E. F. Iannetti, J. Smeitink, and J. Beyrath, Multiplexed high-content analysis of mitochondrial morphofunction using live-cell microscopy, Nat Protoc, vol.11, pp.1693-1710, 2016.

A. P. Leonard, R. B. Cameron, and J. L. Speiser, Quantitative analysis of mitochondrial morphology and membrane potential in living cells using high-content imaging, machine learning, and morphological binning, Biochim Biophys Acta, vol.1853, pp.348-360, 2015.

J. Zhang, E. Nuebel, and D. Wisidagama, Measuring energy metabolism in cultured cells, including human pluripotent stem cells and differentiated cells, Nat Protoc, vol.7, pp.1068-1085, 2012.

M. Boutros, F. Heigwer, and C. Laufer, Microscopy-Based High-Content Screening, Cell, vol.163, pp.1314-1325, 2015.

M. A. Horwitz, Formation of a novel phagosome by the Legionnaires' disease bacterium (Legionella pneumophila) in human monocytes, J Exp Med, vol.158, pp.1319-1331, 1983.

A. Tiaden, T. Spirig, and S. S. Weber, The Legionella pneumophila response regulator LqsR promotes host cell interactions as an element of the virulence regulatory network controlled by RpoS and LetA, Cellular Microbiology, vol.9, pp.2903-2920, 2007.