75 DLL “Plan-Apochromat” or a 100×/1.4 DIC objective. C59 wnt cost Metamorph 7.5 (Molecular Devices) or NIS-Element (Nikon) software was used for digital analysis and treatment of the images to extract the

number, specific fluorescence intensity and length of stained bacteria. This system allows enumeration and manual differentiation between individually labeled cells, cells in aggregations and/or auto fluorescent particles which can interfere with automated analysis. For the CV6 procedure, cells were scored as viable if their fluorescence intensity was at least 1.5 times greater than the fluorescence background noise. Under our conditions, detection limits for CV6 measurement were 3 × 104 cells ml-1. Co-culture of L. pneumophila and A. Castellanii Axenic cultures of A. JAK inhibitor castellanii (ATCC 30234) were Momelotinib prepared as previously described [48]. Briefly,

the A. castellanii strain was grown in a 150-cm2 cell culture flask in PYG medium (peptone-yeast extract-glucose) at 30°C for 3 days. Monolayers were developed in 24-well tissue culture plates using Page’s amoeba Saline (PAS) for 24 h at 30°C. Aliquots of 1 × 106 amoebae per well in 24-well tissue culture plates were infected with 10 × 1 ml of L. pneumophila at 1 × 108 cells ml-1 in PAS as described above (MOI 100). The plates were centrifuged at 500 × g for 5 min and incubated for 3 days at 37°C. Then, the monolayer and supernatant were removed and spread on BCYE agar plates. Colonies were counted after 3 days and 10 days of incubation at 37°C. Acknowledgement We thank Gail G. Hardy (Indiana university, Bloomington), Audrey Dumont (CNRS, Marseille), Emilie Fugier (CNRS, Marseille), and Sophie Jarraud (CNRL, France) for discussions and critical reading of the manuscript. This work was supported by a fund from the ANSES (Program ARCL-2005) and a doctoral fellowship (Adrien Ducret) from CIBA

SA. We also thank Yannick Fovet for his helpful comments on the manuscript. We thank Bernard Lascola and Isabelle Pagnier for their generous gifts of LP1 and amoebae. References 1. Fliermans: Ecology of legionella: from data to knowledge Amino acid with a little wisdom. Microb Ecol 1996, 32:203–228.PubMedCrossRef 2. Muldrow LL, Tyndall RL, Fliermans CB: Application of flow cytometry to studies of pathogenic free-living amoebae. Appl Environ Microbiol 1982, 44:1258–1269.PubMedCentralPubMed 3. Fields BS, Benson RF, Besser RE: Legionella and legionnaires’ disease: 25 years of investigation. Clin Microbiol Rev 2002, 15:506–526.PubMedCentralPubMedCrossRef 4. Fields BS: The molecular ecology of legionellae. Trends Microbiol 1996, 4:286–290.PubMedCrossRef 5. Molmeret M, Horn M, Wagner M, Santic M, Abu Kwaik Y: Amoebae as training grounds for intracellular bacterial pathogens. Appl Environ Microbiol 2005, 71:20–28.PubMedCentralPubMedCrossRef 6. Newton HJ, Ang DKY, van Driel IR, Hartland EL: Molecular pathogenesis of infections caused by legionella pneumophila. Clin Microbiol Rev 2010, 23:274–298.