Glycerol

Recombinant strains of Mtb H37Rv expressing an enhanced green fluorescent protein (GFP) or a red fluorescent protein DsRed [49 (link)] were cultured in Middlebrook 7H9 medium (Difco) supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC, Difco), 0.2% glycerol (Euromedex), 0.05% Tween 80 (Sigma-Aldrich) and 50 μg/ml hygromycin (ThermoFisher Scientific) or 25 μg/ml kanamycin (Sigma-Aldrich) for H37Rv-GFP or H37Rv-DsRed, respectively. Cultures were maintained for 14 days until the exponential phase was reached. Before cell infection, bacilli were washed with Dulbecco’s Phosphate Buffered Saline (DPBS, free from MgCl2 and CaCl2, Gibco), resuspended in 10 mL RPMI-FBS and centrifuged at 1000 RPM for 2 min at room temperature to remove bacterial aggregates. Bacterial titer of the suspension was determined by measuring the optical density (OD600 nm) and GFP or DsRed fluorescence on a Victor Multilabel Counter (Perkin Elmer). The bacterial suspension was diluted at the required titre in RPMI 1640 supplemented with 10% FBS prior to infection. For in vivo studies, the non-fluorescent Mtb H37Rv strain were grown in Middlebrook 7H9 medium, as described previously [50 (link),51 (link)].