Mouse il 17 elispot kit

10 days after EAE induction, the fraction of cytokine-producing cells among total splenocytes was determined, either by using a mouse IL-17 ELISpot kit (R&D Systems) according to the manufacturer’s protocol, or by performing IFN-γ ELISpot assays as described previously (Stern et al., 2010 (link)). All ELISpots assay plates were counted using an Immunospot™ counter.

Eight or nine days after the second booster, mice (3–4 mice/group) were euthanized with Pentobarbitone sodium (100–150 mg/kg, i.p.) and spleens were harvested, homogenised and forced through 70 μm cell strainer (BD) with the ends of sterile syringe plungers [45 (link)]. The splenocytes were obtained by centrifugation at 250 g. Erythrocytes were lysed with ACK buffer for 5 min at room temperature. After washing once with PBS, splenocytes were resuspended in RPMI 1640 medium with 10% FBS, and filtered with 70 μm cell strainer (BD) to get rid of cell aggregates.
Interferon-gamma (IFN-γ) and Interleukine-17A (IL-17A) ELISPOT assays were conducted with Mouse IFN-γ ELISPOT kit (R&D Systems) and Mouse IL-17 ELISpot Kit (R&D Systems) respectively, according to the manufacturer’s instructions. Briefly, splenocytes, stimulated by antigen rSaEsxA or rSaEsxB at the concentration of 2 μg/mL, were plated at the concentration of 5E + 05 cells/well in duplicate for 20 h at 37 °C. Then plates were washed and incubated with biotinylated anti-IFN-γ or anti-IL-17A antibody overnight at 4 °C. After washing the plates, streptavidin-Alkaline phosphatase was added and incubated for 2 h at room temperature. At last, the plates were incubated with substrate BCIP/NBT Chromogen for 0.5-1 h at room temperature for color development. The spots were counted using an immunospot reader system.