96-well strip precoated elispot plate  (Mabtech Inc)

Journal: bioRxiv

Article Title: Sez6L2 autoimmunity induces cerebellar ataxia in mice

doi: 10.1101/2025.05.28.656724

Figure Lengend Snippet: A and B) Splenocyte/Lymph node mixed cell cultures from Sez6L2 or sham immunized mice were left unstimulated or were stimulated with H-Sez6L2 or M-Sez6L2 protein as indicated for 24 hours in an IFNγ ELISPOT assay. N=8-10. A) Representative images of ELISPOT wells. B) Quantification of the number of spots per 200,000 splenocytes/lymph node cells plated. C-E) Splenocyte/Lymph node mixed cell cultures from Sez6L2 or sham immunized mice were stimulated with M-Sez6L2 protein for 24 or 72 hours followed by cell surface and intracellular cytokine staining and analysis by flow cytometry. C) Graphs show the percent of CD4 + cells positive for IFNγ or TNFα after stimulation in culture for 24 hours. N=8-10. D) Graphs show the percent of CD4 + cells positive for IFNγ, TNFα, IL-4, or IL-17A after stimulation in culture for 72 hours. M=4-5. E) Graphs show the percent of CD8 + cells positive for IFNγ, TNFα after stimulation in culture for 24 hours. N= 8-10. Statistics for all graphs = Welch-ANOVAs with Dunnett’s T3 MCTs.

Article Snippet: Mixed splenocyte and lymph node cells were plated on IFNγ ELISPOT pre-coated plates (Cellular Technology Limited, mIFNgp-2M) in serum-free media at a cell density of 200,000 cells per well with or without 8 μg/mL mouse or human recombinant Sez6L2 protein.

Techniques: Enzyme-linked Immunospot, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: Sez6L2 autoimmunity induces cerebellar ataxia in mice

doi: 10.1101/2025.05.28.656724

Figure Lengend Snippet: A & B) Motor functions assessed at 5.5 weeks post-immunization on a mixed-sex cohort. Sham N=8; H-Sez6L2 N=9. A) Foot faults per minute of mobile activity on a wire grid. B) Time to descend a metal pole. Statistical tests: t test. C and D) Splenocytes from H-Sez6L2 or sham-immunized mice were stimulated with recombinant mouse Sez6L2 protein (C) or a 15mer/10aa overlapping peptide library containing peptides from the extracellular domain of mouse and human Sez6L2 (D) and assayed in an IFNγ ELISPOT assay. In D, the 15mer immunodominant peptide sequences are shown with the MHC-II core binding region predicted by IEDB Resource highlighted in blue. For the peptide ELISPOT, N=5 Sez6L2-immunized and N=3 for sham.

Article Snippet: Mixed splenocyte and lymph node cells were plated on IFNγ ELISPOT pre-coated plates (Cellular Technology Limited, mIFNgp-2M) in serum-free media at a cell density of 200,000 cells per well with or without 8 μg/mL mouse or human recombinant Sez6L2 protein.

Techniques: Activity Assay, Recombinant, Enzyme-linked Immunospot, Binding Assay