When indicated, 10 mM 3-methyladenine (Sigma, directly prepared in PLX-4720 mouse DMEM medium) was added to the cell monolayers to inhibit autophagy prior to infection. To investigate the presence of Brucella in LC3B-positive autophagosomes, we established stable clones of MEFs expressing GFP-LC3 WT (plasmid pEX-GFP-hLC3WT, Addgene). Starvation-induced autophagy was obtained by a 2 h-incubation in EBSS medium (Earle’s Balanced Salt solution) after three washes

with PBS to remove serum. Cell infection with Brucella Growth of bacteria was assessed by measuring the optical density (OD) at a wavelength of 600 nm considering that an OD = 1 corresponds to 1×109 bacteria/mL. Then, bacteria were sedimented by centrifugation at 900 g for 10 min to discard 2YT medium and resuspended in the same volume of DMEM + 10% FCS. After dilution of the bacterial suspension in an appropriate volume of DMEM + FCS to get an MOI (multiplicity of infection) of 300, RAD001 in vitro the culture medium present in 12-well plates containing MEFs was withdrawn and replaced by the bacterial suspension. The Petri dishes were centrifuged for 10 min at 400 g at 4°C to favour the adhesion of bacteria to the cell surface and then placed

in a 5% CO2 incubator at 37°C (this is the time zero postinfection). The passage from 4°C to 37°C aims at synchronizing the entry of bacteria into the cells. After one hour of infection, wells were washed thrice with sterile phosphate-buffered saline (PBS, 136.9 mM NaCl, 2.7 mM KCl, 10.1 mM Na2HPO4 and 1.8 mM KH2PO4) and further incubated for one hour with DMEM + FCS containing 50 μg of gentamicin per mL to kill extracellular Histidine ammonia-lyase bacteria. Afterwards, the medium was changed and replaced by the medium containing 10 μg of gentamicin per mL until the end of the postinfection period [28]. For the counting of viable intracellular bacteria using colony forming units (CFUs), after

infection with Brucella, cells were washed thrice with PBS then lysed for 10 min at room selleck screening library temperature in 800 μl of PBS containing 0.1% Triton X-100 under manual agitation. Lysates were diluted from 10 to 1,000 times in PBS and plated on Petri dishes containing 2YT Agar. Petri dishes were incubated for three to four days at 37°C before the counting of colony forming units. Fluorescence microscopy To count the number of Brucella per infected cell, we infected MEFs with Brucella-mCherry. At various time points p.i., cells were washed twice with filtered dPBS (PBS supplemented with 0.88 mM CaCl2 and 0.5 mM MgCl2), fixed for 20 min at room temperature in 4% paraformaldehyde in cold PBS, then washed thrice with dPBS. Nuclei were stained with 4’-6-diamidino-2-phenylindole (DAPI) prepared in PBS containing 0.1% Triton X-100 and washed three times with PBS. Coverslips were mounted in Mowiol on glass plates. Fluorescence was observed using a Nikon i80 fluorescence microscope. In an attempt to detect Brucella in compartments stained with LC3, we infected cells expressing GFP-LC3 with B.