Rifampicin Susceptibility Assay with AF488

The AF488 assay was adapted from reference^{9 (link)} with some modifications. SNAP-Surface Alexa Fluor 488 (NEB) was dissolved in DMSO for 5 mg/ml stock solution and stored at −80 °C for up to several weeks. 2 ml of the strain to be tested was cultured in 7H9 medium until exponential growth phase with optical density (OD_600nm = 0.6–0.8). 1 ml bacteria were inoculated in 7H9 medium with and without 1 μg/ml rifampicin respectively for 3 hours. After two washes with1ml PBST, bacteria was re-suspended in 50 μl diluted AF488 (working concentration 200 μg/ml). After incubating in the dark at room temperature for 5 min, bacteria were transferred into a fresh tube and then washed twice more with 1 ml PBST. Bacterial pellets were re-suspended in 500 μl 7H9 fresh medium. 100 μl of the suspension were inoculated into 7H9 medium containing 0 μg/ml, 10 μg/ml, 50 μg/ml rifampicin. After 16 hours culture in 37 °C shaking incubator in the dark, 100ul samples were fixed by 100ul 4% Paraformaldehyde (PFA) at room temperature for 20 min. BD Accuri C6 desktop flow cytometry was used to collect fluorescence intensity with 488 nm excitation laser and 533 nm emission filter. % Dim cells representing the relative percentage of growing cells were analyzed by Flow-Jo software.