Macrophage infection with M. tuberculosis

THP-1 macrophages were exposed to CS or e-vapor extract, and then infected with M. tuberculosis following a protocol previously reported [33], with some modifications. Briefly, 3 × 10⁵ cells per well were PMA stimulated and seeded in 24-well tissue culture plates with clear bottoms (Falcon^®). Freshly prepared 10% CS extract or 100% e-vapor extract were added to the cells and incubated for 3 hours for CS extract and 24 hours for e-vapor extract at 37°C in 5% CO₂. For the infection, mid-log phase M. tuberculosis were washed twice with DPBS+ 0.05% tween and subsequently once with DPBS after which they stood for 5 min, before the supernatant were collected. The bacteria were then diluted in RPMI with CS extract or e-vapor extract and added to the THP-1 macrophages at a multiplicity of infection (MOI) of 0.1. After 3 h of contact at 37°C in 5% CO₂, the macrophages were treated with 200 μg/mL amikacin for 1 h and washed three times with DPBS to eliminate any extracellular bacteria. Lastly, 1 mL of RPMI with CS extract or e-vapor extract was added to each well and incubated at 37°C in 5% CO₂. Fresh medium with CS extract or e-vapor extract was added at day 3. Intracellular growth was assessed by lysis of the monolayers by the addition of 500 μL of water followed by a 30 min incubation at room temperature and serial dilution in PBS-tween plating onto Middlebrook 7H10 solid medium at days 0, 1, 2, 3 and 6. Colonies were counted after 3–4 weeks incubation at 37°C and the average CFUs/mL determined.