Series 2 immunospot analyzer

Cytokine ELISPOT assay was performed and spots analyzed as described previously (18 (link), 19 (link)). In brief, ELISPOT plates (Millipore, Billerica, MA, USA) were precoated with anti-mouse-IFN-γ mAb (eBioscience, AN-18) and anti-mouse-IL-17 mAb (Bio X Cell; 17F3). Splenocytes (5 × 10⁵ cells/well) and brain-infiltrating mononuclear cells (2–5 × 10⁴ cells/well) were restimulated with MOG_35–55 peptide in HL-1 medium (Lonza) at 37°C for 24 h. Biotinylated anti-mouse-IFN-γ mAb (eBioscience; R4-6A2) and anti-mouse-IL-17 mAb (BioLegend, TC11-8H4) were then added overnight at 4°C, followed by incubation with streptavidin alkaline phosphatase (Invitrogen, Waltham, MA, USA) for 2 h at room temperature and developing with BCIP/NBT Phosphatase Substrate (KPL, Gaithersburg, MD, USA). After plate developing, image analysis of spots was performed on a Series 2 Immunospot analyzer (Cellular Technology Limited, Cleveland, OH, USA). Results for antigen-specific spot-forming cells were normalized with a negative control containing peptide-free media. All measurements were performed in duplicate or triplicate.

Cytokine ELISPOT assay was performed and spots were analyzed as described previously [18 (link),19 (link)]. In brief, ELISPOT plates (Millipore, Billerica, MA, USA) were precoated with anti-mouse-IFN-γ mAb (eBioscience, San Diego, CA, USA, AN-18) and anti-mouse-IL-17 mAb (17F3, Bio X Cell, Lebanon, NH, USA). Splenocytes (5 × 10⁵ cells/well) were restimulated with MOG35–55 peptide in HL-1 medium (Lonza, Basel, Switzaerland) at 37 °C, 5% CO₂ for 24 h. Biotinylated anti-mouse-IFN-γ mAb (R4-6A2, eBioscience/Thermo Fisher, Waltham, MA, USA) and anti-mouse-IL-17 mAb (TC11-8H4, BioLegend, San Diego, CA, USA) were then added overnight at 4 °C, followed by incubation with streptavidin alkaline phosphatase (Invitrogen, Waltham, MA, USA) for 2 h at room temperature and developing with BCIP/NBT Phosphatase Substrate (KPL, Gaithersburg, MD, USA). After plate development, image analysis of spots was performed on a Series 2 ImmunoSpot analyzer (Cellular Technology Limited, Cleveland, OH, USA). Results for antigen-specific spot-forming cells were normalized with a negative control containing peptide-free media. All measurements were performed in triplicate.