Intracellular Mycobacterium tuberculosis Infection

Mtb [DsRed-expressing H37Rv (24 (link))] was cultured in Difco Middlebrook 7H9 broth (Becton Dickinson, Breda, The Netherlands) supplemented with 10% ADC (Becton Dickinson) and 0.05% Tween 80 (Sigma-Aldrich). One day before infection, Mtb cultures were diluted to a density corresponding with early log-phase growth (OD₆₀₀ of 0.25). The following day, bacterial suspensions (or 7H9 for mock infections) were diluted in cell culture medium without antibiotics to reach a multiplicity of infection (MOI) of 10. MOI of the inoculum was verified by a standard colony-forming unit (CFU) assay. Cells seeded in 96-well flat-bottom plates at a density of 30,000 cells/well in appropriate cell culture medium without antibiotics 1 day prior to infection, were inoculated with 100 μl of the bacterial suspension, centrifuged for 3 min at 800 rpm, and incubated at 37°C/5% CO₂ for 60 min. Bacteria were then washed away with cell culture medium containing 30 μg/ml gentamicin sulfate (Lonza BioWhittaker, Basel, Switzerland), incubated for 10 min at 37°C/5% CO₂, followed by replacement with medium containing 5 μg/ml gentamicin sulfate and, if indicated, chemical compounds until readout by flow cytometry, Luminex, or CFU.