Macrophage Infection by Mycobacteria

The human monocytic cell line THP-1 (ATCC TIB 202) was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco, Invitrogen, Saint Aubin, France) at 37 °C with 5% CO₂. The cells were transferred to a 24-well plate at 1.5 × 10⁵ cells per well and pretreated with 100 µg/mL phorbol 12-myristate 13-acetate for 48 h to induce differentiation into macrophages. Mycobacteria (M. shigaense, M. triplex and M. tuberculosis) were added to the macrophage culture in triplicate wells at a multiplicity of infection (MOI) of 1 in order to generate a detectable immune response^{22 (link)}. A mock infection was performed with culture medium. After 6 h at 37 °C and 5% CO₂, the infected macrophages were washed with 1 × Hanks solution (Gibco) to remove extracellular mycobacteria and then incubated with fresh medium. At 6, 24, and 48 h, the cells were lysed with 0.05% Tween-20 (Sigma Aldrich) and plated on Löwenstein–Jensen (L–J) medium. Colony forming units (CFUs) from the infected cells were compared with those of the original inoculum size of mycobacteria plated directly on L–J medium. The entire experiment was repeated thrice, thereby yielding a total of nine wells that included three wells per species, including mock-infected cells.