M.tb H37Rv culture

M.tb H37Rv strain (ATCC 27294) was acquired from the American Type Culture Collection. M.tb H37Rv was cultured in Middlebrook 7H10 medium supplemented with 10% OADC, 0.5% glycerol, 0.05% Tween-80, and 10 μg/ml amphotericin in a 37°C incubator with 10% CO₂. Cultures were harvested in midlog phase and frozen down in 15% glycerol. To standardize infection doses, cells were infected from frozen stocks, and multiplicity of infections (MOIs) were determined based on the mycobacterial counts of the stocks. For infections, M.tb bacilli were diluted in cell culture medium, washed by centrifugation, and taken up in cell culture medium for infection experiments. M.tb was sonicated for 40 s in an ultrasonic water bath to disperse clumps before inoculation of cell culture. M.tb bacilli were added onto WI-38 cells for the interval time. Before cell lysis, 200 μg/ml amikacin was added for 2 h. The cell lysates were analyzed using the Western blotting method.