2.5. Tumor Growth Inhibition Experiments

Growth inhibition experiments were performed as previously described [8,30,31]. Briefly, M. brumae or M. bovis BCG cells were taken in sterile conditions with a Kollë handle from the bottom part of the pellicle to the top. Then, mycobacteria were disaggregated with glass beads and a suspension similar to McFarland 1 standard was performed. Bacteria concentration was corroborated by culturing in Middlebrook 7H10 medium for CFU counts (Supplementary Table S2). Tumor cells (3 × 10⁴) were seeded into 96-well tissue culture plates (Nunc) and infected with M. bovis BCG or M. brumae (MOI 10) grown in different culture media. Three hours later, extracellular mycobacteria were removed by washing with warm PBS. Complete medium was then added, and cells were incubated for 72 h. After incubation, supernatants were collected, centrifuged and stored at −80 °C for further cytokine analysis. Tumor cell viability was then performed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Sigma-Aldrich, St. Louis, USA).