Microplate alamarBlue assays (MABAs).

Logarithmically growing M. tuberculosis was inoculated into 7H9 medium in 96-well plates with wells containing increasing concentrations of compound. M. tuberculosis was inoculated at an ODλ₆₀₀ of 0.0008, corresponding to approximately 4 × 10⁵ CFU/ml in 200 μl per well. The plates were incubated at 37°C in 5% CO₂ for 1 week, at which point 32.5 μl of a mixture containing an 8:5 ratio of 0.6 mM resazurin (Sigma) dissolved in 1× phosphate-buffered saline to 20% Tween 80 was added, and the production of fluorescent resorufin was measured after incubation at 37°C in 5% CO₂ overnight. For M. tuberculosis, samples were removed from the plate and mixed with formalin to kill the M. tuberculosis bacteria before measuring the fluorescence. For M. smegmatis, the assay plate was measured directly. Fluorescence was measured on a Tecan M200 Pro plate reader with an excitation λ of 530 nm and an emission λ of 590 nm. For each assay, medium alone served as a negative control, and untreated M. tuberculosis or M. smegmatis was included as a positive control. The percent inhibition was calculated as the {[(fluorescence of the positive control − fluorescence of the negative control) − (fluorescence of the sample − fluorescence of the negative control)]/(fluorescence of the positive control − fluorescence of the negative control)} × 100.