**2.2 Experimental model of airways-induced pulmonary tuberculosis in mice**

*M. tuberculosis* strains were grown in Middlebrook 7H9 medium (Difco Laboratories) supplemented with OADC (Difco Laboratories). After 1 month of culture, mycobacteria were harvested, adjusted to 2.5x105 bacteria in 100µl sterile endotoxin-free saline solution, aliquoted, and maintained at -70ºC until used. Before use, bacteria were stained with fluorescein diacetate (InvitroGen, F1303) and viable bacteria (Kvach, J. T. and Veras, J. R. 1982) (green fluorescence) were counted with an epifluorescence microscope and adjusted to the infective dose. We used the murine model of intra-tracheal infection as described previously (Hernandez-Pando, R. 1996), with some modifications.

Briefly, 3-5 male BALB/c mice from 6-8 weeks of age were anaesthetized with sevoflurane, and 100 μl isotonic sterile endotoxin-free saline solution with 2.5 x 105 viable bacilli were inoculated intra-tracheally. Control animals were inoculated only with isotonic, sterile endotoxin-free saline solution without bacilli. Animals were then maintained in cages fitted with microisolators in a P-3 biosecurity level facility. The protocol was institutionally approved according to ethical norms for use of animals in experimentation. Following infection, at least three to five mice per group were euthanized at every time point selected for the various analysis.
