Concanavalin cona

Male mice, either wild type or PPARα^−/−, were administered Concanavalin (ConA; Sigma, St. Louis, MO) at a dose of 15 mg/kg in sterile saline via tail vein injection as has previously been described [21 (link)]. Mice were then anesthetized with ketamine and xylazine (100 and 10 mg/kg respectively) 10 or 24 h following injection, the diaphragm severed to effect euthanasia, and serum and tissue collected.

The spleens of the adult offspring rats in two groups were aseptically removed and splenic lymphocytes were isolated by standard Ficoll–Paque density gradient from each animal. The cells (1 × 10⁵/ml) were co-cultured with either 100 ng/ml SEB or 5 μg/ml concanavalin (Con) A (Sigma-Aldrich, St Louis, MO) for 3 days in 96-well and 24-well culture plates in 200 μl RPMI medium (Gibco BRL, USA) containing 10% FBS, L-glutamine, penicillin and streptomycin in a humidified incubator in 5% CO2 at 37 °C. For analysis of the T cells subpopulation, the lymphocytes in 24-well culture plates were harvested at the end of culture days 1, 2, & 3 and stained for the analysis of T cell subpopulation with flow cytometry as shown below. For the experiment of the lymphocyte proliferation, 1 μCi ³H-thymidine was added to the lymphocytes in 96-well culture plates 5 h before the end of culture days 1, 2, & 3. Thymidine incorporation by cells was determined using a cell harvester and 1450 MICROBETA liquid scintillation counter (PerkinElmer®, Waltham, MA, USA). Proliferation was measured as radioactivity incorporation [presented as counts per minute (CPM)].

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.