In Vitro Infection of Alveolar Macrophages and Lung Epithelial Cells with Brucella abortus

Alveolar macrophages (AM) and LEC were infected in vitro with B. abortus at multiplicities of infection (MOI) of 100 bacteria/cell (AM) or 200 bacteria/cell (LEC) as described previously (14 (link)). Bacteria were dispensed suspended in culture medium without antibiotics, followed by centrifugation of the plates and incubation for 2 hours (37°C, 5% CO₂ atmosphere). Bacteria not adhered or internalized into the cells were removed by three washes with sterile PBS (time 0 p.i.). To kill non-internalized bacteria the cells were incubated in culture medium containing 100 µg/ml of gentamicin (Sigma, USA) and 50 µg/ml of streptomycin (Sigma, USA). Culture supernatants were harvested for cytokine measurement 24 h after antibiotics addition. At the same time, cell lysates prepared using 0.2% Triton X100 were plated on TSA to enumerate CFU of intracellular bacteria. In some experiments, a STING inhibitor (H151, InvivoGen, 15 µM) or its vehicle (DMSO) were added to AM and LEC for 24 h before infection, and were maintained during the whole infection period.