Bovine in vitro cytokine response

A total of 500 mL of heparinized blood samples was cultured in triplicate within two hours of collection in 24-well plates (Thermo Fisher Scientific, USA) with 500 mL RPMI supplemented with 10% foetal bovine serum (Thermo Fisher Scientific, Waltham, USA) and 1% penicillin-streptomycin-amphotericin B mixture (Lonza, Belgium) referred to as complete medium (CM), as described previously (Vázquez et al., 2019 (link)). Each blood replicate was also supplemented with N. caninum soluble extract antigen obtained from Nc-Spain7 tachyzoites (Alvarez-García et al., 2003 (link)) at 5 μg/mL, concanavalin (ConA) (Sigma-Aldrich, Spain) at 5 μg/mL as a positive control, and PBS as negative control. After 24 h of incubation (37°C, 5% CO₂), culture supernatants were collected by centrifugation of plates at 1000 × g for 10 min at 4°C and stored at −80 °C until laboratory analysis for the evaluation of IFN-γ and IL-4 release.
Cytokine release was measured with commercial bovine IFN-γ and bovine IL-4 ELISA kits (Mabtech AB, Nacka Strand, Sweden) following the manufacturer’s guidelines. The cytokine concentrations were calculated by interpolation from a standard curve produced with recombinant cytokines provided with the kits. The colour reaction was developed by the addition of 3,3’,5,5’-tetramethylbenzidine substrate (TMB, Sigma-Aldrich, Spain). Plates were read at 450 nm.