Canine ifn γ elispot kit

PBMCs were isolated using Ficoll-Paque Plus (GE Healthcare, Piscataway, NJ). Briefly, 5 ml of EDTA-treated whole blood was diluted 1:2 with RPMI 1640 medium (Gibco, Gaithersburg, MD) at room temperature, layered slowly over 5 ml Ficoll-Paque Plus in a 15-ml tube, and centrifuged at 400 × g for 30 min at 20°C. The PBMC layer was collected and washed twice with RPMI 1640 (along with centrifugation at 350 × g for 5 min), and PBMCs were resuspended in 1 ml RPMI 1640 containing 10% fetal bovine serum and a mixture of 1% l-glutamine and 1% antibiotic (Gibco). The ELISpot assay was performed using the canine IFN-γ ELISpot kit (R&D Systems, Minneapolis, MN). Briefly, 5 × 10⁵ PBMCs/well were plated in a canine IFN-γ-coated 96-well plate. The cells were stimulated with rEtpE-C (1 μg) or culture medium as a negative control. The immunopositivity of spots was assessed with an ImmunoSpot analyzer (Cellular Technology, Shaker Heights, OH).

Four hundred thousand PBMCs were cultured in media, 2 ng/mL phorbol 12-myristate 13-acetate (PMA) and 500 ng/mL Ca²⁺ ionomycin (both Sigma-Aldrich, St. Louis, MO, USA), or 10 μg/mL T. cruzi amastigote lysate for 16 h at 37 °C and 5% CO₂. Cells were assayed for IFNγ production using the Canine IFN-γ ELISpot kit (cat no. EL781, R& D Systems, Inc, Minneapolis, MN, USA) following standard kit protocol. Enumeration of spot forming cells was completed by an ImmunoSpot analyzer (CTL, Cleveland, OH, USA). Mean numbers of spots from duplicate or triplicate well were obtained for media and T. cruzi lysate stimulation conditions. Responses were considered positive if a minimum of 20 spots/10⁶ PBMC total were present and this number was at least twice the value of wells assayed with media alone [27 (link)].

PBMCs were isolated using Ficoll-Paque Plus (GE Healthcare, Piscataway, NJ) as previously described (22 (link)). The ELISpot assay was performed using the Canine IFN-γ ELISpot kit (R&D Systems, Minneapolis, MN). Briefly, 5 × 10⁵ PBMCs/well were plated in a 96-well plate coated with canine IFN-γ. The cells were stimulated with rOMP-1B (1 μg), rVirB2-4 (1 μg), or culture medium (as a negative control) in triplicate or duplicate wells per dog. The immunopositivity of spots was assessed with an ImmunoSpot S6 Analyzer (Cellular Technology, Cleveland, OH). The data were analyzed by a negative binomial mixed model (22 (link), 89 (link)).