To infect the monocyte-derived cells, dilute the bacteria to a 5 times 10 to the sixth colony-forming units per milliliter concentration in serum-free medium, and replace the supernatant in each well of the six-well cell culture plate, with 1 milliliter of fresh serum-free medium and 1 milliliter of bacteria suspension per well to obtain a multiplicity of infection of 5.
After a 4-hour incubation in the cell-culture incubator, wash each well three times with 1 milliliter of sterile wash buffer per wash, tilting the plate carefully to remove the entire volume of buffer from the corners of the wells after each wash. Then, resuspend the MTb-infected monocyte-derived cells in 2 milliliters of complete medium without antibiotics.
