Human t helper cytokine panel legendplex bead based immunoassay

Cytokines directly in the CSF were measured using the Human T Helper Cytokine Panel LEGENDplex bead-based immunoassay (BioLegend) according to the manufacturer's instructions.
Intrathecal neurofilament light chain (NF-L) and chitinase 3-like 1 (CHI3L1) proteins were quantified in CSF samples by ELISA (Human Diagnostics, Umea, Sweden, and MicroVue, Athens, OH, respectively) according to the manufacturer's instructions.

Supernatants were collected from the wells of cultured CFSE-labeled PBMCs after 7 days. Cytokines were measured with Human T Helper Cytokine Panel LEGENDplex bead-based immunoassay (Biolegend; incl. measurement of 13 human cytokines: IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21, IL-22, IFN-γ and TNF) using flow cytometry according to the manufacturer's instructions. IFN-γ ELISA (Biolegend) and GM-CSF ELISA (BD Biosciences) was performed according to the manufacturer's instructions. Heatmaps for cytokine responses were generated using Spice software (NIH, NIAID). For visualization purposes in graphs using a log axis absent values or values below standard range were set as 1.

Release of interferon gamma (IFN-γ) was measured using the ELISA MAX Deluxe Set Human IFN-γ (BioLegend, San Diego, CA) according to the manufacturer's instructions. A well was considered positive if IFN-γ ≥20 pg/mL, and a patient was considered positive for a peptide if the mean IFN-γ of the four wells was ≥20 pg/mL and at least 3 of the 4 wells were positive.
Cytokines in supernatants and CSF were measured using the Human T Helper Cytokine Panel LEGENDplex bead-based immunoassay (BioLegend) according to the manufacturer's instructions.