Preparation and infection of murine bone marrow-derived macrophages.

Femurs and tibias of female C57BL/6 mice were extracted, and bone marrow cells were aseptically flushed using PBS. Cells were resuspended in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum (FBS), 10 mM HEPES, and 20% (vol/vol) L929 culture filtrate (LCM) and incubated for 6 days to allow differentiation into macrophages. Cells were harvested and seeded at 6 × 10⁴ cells per well in 96-well plates in 10% LCM complete DMEM overnight before infection. Macrophages were activated with IFN-γ (20 ng/ml) overnight before infection and throughout the infection. Mycobacteria were washed in PBS + 0.05% tyloxapol, and a single cell suspension was generated by low-speed centrifugation to pellet clumped cells. The bacteria were diluted into 10% LCM and added to macrophages at a multiplicity of infection (MOI) of 0.1. After 4 h, extracellular bacteria were removed by washing the macrophages three times with warm PBS. The number of intracellular bacteria was determined by lysing macrophages with 0.01% Triton X-100 and culturing serial dilutions of macrophage lysates on 7H10 agar plates.