Infection of Macrophages with Fluorescent M. tuberculosis

M. tuberculosis was grown to log phase in 7H9 liquid media (BD 271310) supplemented with Middlebrook OADC (Sigma M0678), 0.5% glycerol, 0.05% Tween-80 in roller bottles at 37°C. M. tuberculosis Erdman strain expressing eGFP under control of the MOP promoter was a gift from Dr. Sarah Stanley’s laboratory. Macrophages were infected with fluorescent M. tuberculosis using a modified version of the spinfection protocol as previously described (Watson et al., 2015 (link)). Mycobacteria were washed in PBS three times and directly inoculated into the macrophage tissue culture wells at an MOI of 10. Following centrifugation, infected cells were incubated at 37°C for one day post-infection. The wells were washed with PBS and fixed with 4% PFA before staining for microscopy.