Breatheasy

Primary screening was carried out by the Experimental Therapeutics Center (A^*Star, Singapore) using exponentially growing M. bovis BCG diluted to OD₆₀₀ of 0.1 in glycerol-free Middlebrook 7H9 medium and flat bottom clear 384-well plates (Greiner Bio-One) sealed with breathable membranes (Breath Easy, Sigma-Aldrich). Compounds were screened in duplicates at a final concentration of 12.5 μM (40 μl/well) using turbidity as single-point readout after 3 days of incubation at 37°C. The TB first line drug rifampicin at 10 μM served as positive control. All assay wells including control wells had a final concentration of 0.6% DMSO. A pilot screen of 1,000 compounds carried out prior the main campaign confirmed robustness and reproducibility of the assay (signal-to-noise ratio = 5.6 ± 0.3; Z' factor = 0.8 ± 0.1) (Zhang et al., 1999 (link)).

Induced antigen-positive and control cells were seeded at a density of 5000 cells per well into the wells of a 384-well plate in a 12.5 µL complete RPMI medium. Another 12.5 µL volume containing an aliquot of a three-fold serial dilution of antibodies in complete RPMI, starting at 400 nM (200 nM final concentration), was added. The plate was sealed with a semi-permeable membrane (BreathEasy, Sigma Aldrich), and cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂ for 8 days. Proliferation was measured using the CellTiter-Glo^® 2.0 substrate (Promega, Madison, WI, USA). Plates were equilibrated to RT, and 25 µL substrate per well were added. Luminescence was measured immediately using a Spark plate reader (Tecan). The luminescence in wells with untreated cells was considered to correspond to 100% viability, while the luminescence in wells without cells corresponded to the background. Percent viability was calculated as (luminescence in test well/luminescence in wells with untreated cells) × 100 after the background luminescence has been subtracted from all readings. Measurements were performed in triplicate, and the results were processed using GraphPad Prism 5.

Induced antigen-positive and control cells were seeded at a density of 5000 cells per well into the wells of a 384-well plate in a 12.5 µL complete RPMI medium. Another 12.5 µL volume containing an aliquot of a three-fold serial dilution of antibodies in complete RPMI, starting at 400 nM (200 nM final concentration), was added. The plate was sealed with a semi-permeable membrane (BreathEasy, Sigma Aldrich), and cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂ for 8 days. Proliferation was measured using the CellTiter-Glo^® 2.0 substrate (Promega, Madison, WI, USA). Plates were equilibrated to RT, and 25 µL substrate per well were added. Luminescence was measured immediately using a Spark plate reader (Tecan). The luminescence in wells with untreated cells was considered to correspond to 100% viability, while the luminescence in wells without cells corresponded to the background. Percent viability was calculated as (luminescence in test well/luminescence in wells with untreated cells) × 100 after the background luminescence has been subtracted from all readings. Measurements were performed in triplicate, and the results were processed using GraphPad Prism 5.