Cv am

All mycobacteria strains were grown in Middlebrook 7H9 broth (BD, Franklin Lakes, NJ) supplemented with 0.2% (v/v) glycerol, 0.05% (v/v) Tween 80 (MP Biomedicals, Santa Ana, CA), and 10% ADC enrichment (5% bovine serum albumin [Wako Pure Chemical Industries, Osaka, Japan], 0.81% NaCl, and 2% D-glucose) (7H9-ADC broth) or on Mycobacteria 7H11 agar (BD) supplemented with 0.5% (v/v) glycerol and 10% OADC enrichment (ADC enrichment supplemented with 0.06% [v/v] oleic acid) (7H11-OADC agar). For biofilm formation experiment mycobacteria strains were grown on liquid.
Hygromycin B (HYG), kanamycin sulfate (KAN), rifampicin (RMP), amikacin (AMK,) and clarithromycin (CLA) were purchased from Wako Pure Chemical Industries [Osaka, Japan]; and INH from SIGMA-ALDRICH [St. Louis, USA]. AMK stock solutions were prepared in water. Stock solutions of all other compounds were prepared in 100% dimethyl sulfoxide (DMSO) and then filter sterilized (pore size 0.45 µm). These components were frozen in aliquots at − 20 °C.
CV-AM and SG were purchased from Invitrogen (Life-Technologies Corporation, California, USA). CV-AM was dissolved in 250 µl (μl) of DMSO. SG was diluted from the manufacture’s 5 mM stock solution to a final concentration of 50 μM by DMSO. The dyes were stored at − 30 °C.
PMAxx dye was purchased from Cosmo Bio Co., LTD (Tokyo, Japan).

CV-AM (Invitrogen, Life Technologies, USA) is a lipophilic non-fluorescent dye able to permeate the cell membranes of live cells, whereby intracellular esterases cleave the AM group to allow fluorescence. The fluorescent intensity is proportional to the level of functional esterases.
CV-AM was freshly prepared for each experiment by reconstituting a pre-warmed vial in DMSO to prepare a 1 μg/μl working stock. M. tuberculosis from in vitro cultures or recovered from lysed macrophages was stained with 5 ng/µl CV-AM in 500 μl HBSS for 30 min at 37°C with shaking. Samples were fixed in 4% formaldehyde for 30 min, resuspended in HBSS, and transferred to 5 ml polypropylene flow cytometer tubes via a 35 μm cell strainer cap (Corning, USA) for flow cytometric acquisition.
M. tuberculosis::pTiGc was cultured as described above, till OD_600nm = 0.5. Cultures were then inoculated at an OD_600nm = 0.05, following which the OD_600nm was assessed every 2 days for 21 days. Following sampling, an aliquot of culture was resuspended in HBSS to OD_600nm = 0.5 (≈ 5x10⁷ bacteria/ml) and stained with CV-AM to ensure a constant dye:cell distribution. To acquire dead cells for the control sample, 1 ml of cells was heat-killed at 95°C for 60 min, stained with CV-AM and formaldeyhyde-fixed prior to flow cytometric acquisition.