Lysing buffer

Bovine polymorphonuclear neutrophils (PMNs) were isolated from blood taken at the jugular vein in sterile evacuated tubes with EDTA. The tubes were centrifuged (1,000 × g for 10 min at 20°C), the plasma, the buffy coat, and the upper third of the red pellet were removed, and the red blood cells of the remaining pellet were lysed with lysing buffer (Sigma). PMNs were washed once with DPBS without Ca and Mg supplemented with RPMI-AH. The cells were adjusted to 2 × 10⁶/ml. Bacteria were grown overnight in BHI broth. They were washed once in DPBSA+ and resuspended in RPMI-AH at a concentration of 2 × 10⁶ CFU/ml. The phagocytic mixture comprised 250 μl of PMN suspension, 50 μl of bacterial suspension, and various amounts of antisera with or without PCCS as a source of complement. The mixture was adjusted to 0.5 ml with RPMI-AH in 1.5-ml Eppendorf tubes. The tubes were secured on a wheel at 37°C for 90 min and rotated (10 revolutions/min). At the end of the incubation, 50 μl of SDS (0.25% in DPBSA+) was added and the tubes were vortexed. After 1/10 dilution in DPBSA+, the CFU were enumerated by spreading 100 μl of the dilution onto TSA plates.

To prepare splenocytes, single cell suspensions of spleen were treated with lysing buffer (Sigma-Aldrich).
Splenocyte cultures was performed in a 96-well U-bottom plate (Greiner Bio One, Frickenhausen, Germany) at 37°C with 5% CO₂ in GIBCO®RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% FBS (NATOCOR, Carlos Paz, Argentina), 1% GIBCO® GlutaMAX, 100 U/ml penicillin, 100 μg/ml streptomycin (all from Thermo Fisher Scientific) and 50 μM 2-mercaptoethanol (Sigma-Aldrich).
To determine IFN-γ concentration in supernatants, splenocytes (1 × 10⁶ cell/well) were stimulated for 72 h at 37°C with SIINFEKL peptide (1 μg/ml) or OVA (100 μg/ml) and IFN-γ measured by ELISA (IFN-γ ELISA MAX^TMkit, Biolegend, San Diego, CA, USA). The supernatant IFN-γ concentration was calculated after subtraction of background response (cells incubated with media).
To determine intracellular cytokines, splenocytes (3 × 10⁶ cells/well) were stimulated for 5 h with SIINFEKL peptide (2 μg/ml) in the presence of GolgiStop (0.7 μl/ml) and GolgiPlug (1 μl/ml) (BD Bioscience, San Diego, CA, USA). In some experiments, anti-CD107a (ID4B) antibody was added during the incubation with the peptide/GolgiStop/GolgiPlug mix.

Isolated spleens from individual mice were crushed through a sieve (Corning, Durham, USA) and red blood cells were lysed for 5 min in the dark using lysing buffer (Sigma-Aldrich). Cell suspensions were then centrifuged for 5 min at 2000× rpm using a PRISMR centrifuge (Labnet) and cell numbers in the resulting pellet were counted using a cell counting chamber (VWR, Pennsylvania, USA). 1 × 10⁶ splenocytes were plated/well into 48-well culture plates (Costar, Kennebunk, USA) and left either un-stimulated or re-stimulated with 100 µg/ml of stage-specific parasite extract in a total of 800 µl culture medium (RPMI-1640 containing 10% FCS and 0.4% beta-mecaptoethanol; Sigma-Aldrich) at 37 °C and 5% CO₂ for 48 h (cytokines) and 72 h (immunoglobulins). Thereafter, culture supernatants were removed and frozen at − 20 °C until cytokine/chemokine/immunoglobulin levels were determined by Luminex technology.