In Vitro IFNγ Secretion Assay

We analyzed the secretion of IFNγ in vitro following treatment with β-D-glucan with or without inactivated FMDV type O (O PA2) or FMDV type A (A YC) using ELISpot assay kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol. Briefly, isolated murine RPCs or porcine PBMCs (5 × 10⁵ cells/well) were cultured in 96-well polyvinylidene fluoride (PVDF)-backed microplates and treated with 2 μg/mL (final concentration) of inactivated FMDV (O PA2 or A YC) antigen mixed with or without 2.5, 5, 10 μg/mL β-D-glucan for 18 h in an incubator at 37°C with 5% CO₂. PBS and 2 μg/mL of inactivated FMDV type O (O PA2) or type A (A YC) antigen were used as negative control (NC) and positive control (PC), respectively. Data were obtained using the ImmunoSpot ELISpot reader (Autoimmune Diagnostika GmbH, Strassberg, Germany) (4 (link)).