5.6. Intracellular (Macrophage) Drug Screening Assay

Murine J774 cell line was propagated in RPMI 1640 supplemented with l-glutamine and fetal bovine serum (FBS). The cells were maintained in tissue culture flasks at 37 °C in the presence of 5% CO₂. For infection studies, J774 cells were transferred to 12-well tissue culture chambers in 1 mL volumes at a density of 2.0 × 10⁵ in the presence of 10% FBS [25,26]. After overnight incubation, the medium was replaced with fresh medium containing 1% FBS to stop macrophage division while maintaining cell viability. Twenty-four hours later, the macrophage monolayer was enumerated with an ocular micrometer for total number of cells per well to determine the infection ratio. The medium was removed and replaced with 1 mL of fresh medium with 1% FBS containing Mtb at a multiplicity of infection (MOI) of five Mycobacteria/macrophage. The cells were infected for 4 h, after which time nonphagocytosed Mycobacteria were washed from the monolayers and fresh medium was added. Drugs were then added, using three concentrations, and infection was allowed to proceed for 7 days. At 0 and 7 days, the macrophages were lysed with sodium dodecyl sulfate, treated with DNase, diluted, and plated onto 7H10 agar to determine the cell number or colony forming units (CFU). Each drug concentration was tested in duplicate and rifampin was used as the positive control drug. A drug cytotoxicity control plate assay (MTT proliferation) was also conducted in parallel using uninfected macrophages to confirm that concentrations utilized for testing were not toxic to the macrophages.