Confocal microscopy

THP-1 macrophages were seeded on a 12mm circular coverslips in 24-well plate. The infection was performed as described above but using M. bovis BCG-GFP strain or 1-μm-diameter yellow green latex beads (Sigma, St Louis, USA) with a MOI of 10. After infection, cells were fixed overnight with 4% paraformaldehyde (PFA). Coverslips were washed twice with DPBS and incubated with DPBS-BSA 1% 10 min. For staining, coverslips were washed once with DPBS and then 200 μL of DPBS containing Hoechst 33342 (1:1000) and texas red-X-phalloidin (1:200) were added and incubated for 10 min. Hoechst and red-X-phalloidin were purchased in Invitrogen (Waltham, USA). Finally, the coverslips were washed once with DPBS, once with water and subsequently mounted with Prolong Gold Antifade Reagent (Invitrogen). For quantification, 900 cells from three independent experiments (300 cells/coverslip) were counted per day and treatment. The images were taken with an Olympus Fluoview 1000 microscope at days 0, 3 and 6 for the experiments with M. bovis BCG-GFP and at day 0 for the latex beads experiments. The images were analysed using ImageJ software [34]. Latex beads were used in order to determine if phagocytosis was impaired in a general or specific mycobacteria pathway.