1. Mtb infection of monocyte-derived cells

NOTE: The following steps must be performed in a BSL-3 facility.

1. Resuspend the Mycobacterium tuberculosis (Mtb) pellet in serum-free RPMI medium in a new sterile 50 mL tube and adjust the final bacterial concentration to approximately 5 x 10⁶ CFU/mL.
2. Remove the cell culture medium from the 6-well plate(s) containing monocyte-derived cells. Add 1 mL of serum-free RPMI medium to each well. Add 1 mL of bacterial suspension per well to obtain a multiplicity of infection (MOI) 5:1, i.e., 5 x 10⁶ CFU per 10⁶ macrophages in 2 mL/well and incubate the plates for 4 h at 37°C and 5% CO₂.
3. After infection, wash the cells 3 times with 1 mL of sterile wash buffer to remove extracellular bacteria. Tilt the plate and carefully remove the entire wash buffer from the corners. Resuspend the Mtb-infected monocyte-derived cells in 2 mL of RPMI complete medium without antibiotics and proceed to flow cytometry staining or incubate the cells for another 24 h (or other time-points) before flow cytometry.