Bovine PBMC Isolation and Mycobacterium avium Infection

MGIA were performed as previously described (22 (link)–24 (link)). Briefly, fifteen milliliters of peripheral blood were drawn from the tail vein of healthy Holstein cows into heparinized Vacutainer tubes (Becton, Dickinson and Company, Sparks, MD, USA) and diluted 1:2 in Hanks balanced salt solution (HBSS). Leucosep tubes were filled with 15 ml of Ficoll-Paque (1.084 g/cm³) (GE Healthcare, Uppsala, Sweden) and centrifuged at 1,000 rpm for 30 seconds at room temperature. Subsequently, the diluted blood was overlaid on the top of the Ficoll-Paque and centrifuged at 800 g for 15 minutes at room temperature. The plasma layer was removed and the cell interphase containing peripheral blood mononuclear cells (PBMCs) was collected and transferred to a clean tube. PBMCs were washed twice in HBSS and centrifuged at 400 g for 10 minutes to remove platelets. Supernatants were aspirated and the purified PBMCs were resuspended in RPMI-1640 supplemented with 20 mM L-glutamine, 10% heat-inactivated bovine serum (Lonza, Spain), 100 U ml-1 penicillin G, and 100 mg ml-1 streptomycin sulfate (Lonza, Spain). PBMCs were cultured at a concentration of 1 x 10⁶ cells/ml into 24-well plates and incubated at 37°C in a humidified 5% CO₂ incubator for 2 h. Non-adherent cells were removed by washing, and adherent cells were incubated in fresh medium for 7 days at 37°C to allow differentiation to MDMs. Differentiated MDMs were inoculated in triplicate with a single-cell suspension of MAP K10 strain at a multiplicity of infection (MOI) of 10:1 (bacteria:cells). After 2 h, the supernatant was removed, and the cells were washed twice with HBSS to remove extracellular bacteria. Infected MDMs were lysed at this time (2 h p. i.) or were cultured at 37°C for 7 days in fresh medium. At each time point, the supernatant was aspirated and infected MDMs were lysed by vigorous pipetting with 0.5 ml of 0.1% Triton X-100 (Sigma-Aldrich) in sterile water for 10 min.