Long-term PBMC Culture for IFN-γ ELISPOT

Following PBMC isolation, long-term cell culture was performed as described by Maggioli et al.^{39 (link),50} . Briefly, PBMC were cultured (2 × 10⁶ cells/well) in 24 well flat-bottom microtiter plates (Nunc, Thermo Fisher, Waltham, MA) and stimulated with a cocktail of M. bovis PPD (PPD-b, 200 IU/ml, Prionics Ag, Sclieren, Switzerland), rTB10.4 and rAg85A (1 μg/ml each) in cRPMI medium for 12 days 37 °C, 5% CO₂. Media containing human rIL-2 (Sigma, 10 IU/ml) was used to replace media from the PBMC cultures at days 3 and 7. Fresh media without IL-2 was used at days 10 and 12. At day 13, autologous APCs were isolated by adherence incubating 2 × 10⁵/well of freshly isolated PBMC in complete medium at 37 °C, 5% CO₂ for 90 min in 96-well ELISPOT plates (Millipore, Watford, UK) previously coated overnight with anti-bovine IFNγ capture-mAb (Kingfisher) and blocked in cRPMI, for 2 h at 37 °C, 5% CO₂. Non-adherent cells were discarded, and the adherent cells washed twice times with warm cRPMI. Long-term cell cultures and antigen cocktail were added (2 × 10⁴/well) to the ELISPOT plate and incubated for 20 h at 37 °C, 5% CO₂ in the presence of autologous APC.