Elisa max standard set mouse

Mesenteric Lymph Nodes (MLN) were taken from mice during autopsy. They were crushed and filtered (70 µm, BD biosciences) in DMEM (Gibco). Lymphocytes were counted by flow cytometry (Accuri C6, Dutscher) and suspended in DMEM with 100 Unit of Streptomycin, Penicillin (Sigma) and 10% Fetal Calf Serum (FCS) (Eurobio) at a concentration of 2.5 × 10⁵ cells/mL either in 24 wells plate (Costar) pre-incubated with anti-CD3 and anti-CD28 antibodies, 4 µg/mL of each antibody (eBioscience) in PBS or stimulated with PMA ionomycin (eBioscience). Plates were incubated 48 h at 37 °C, 10% of CO2 and cytokine level was assessed by ELISA MAX Standard Set Mouse (BioLegend). The cytokines tested were Th1-related cytokine (IFNγ); Th17-related cytokine (IL17) and Treg– related cytokine (IL10).

Spleens were collected during the remission phase on day 32. Single-cell suspensions were obtained by passing through a 100 µM cell strainer (Becton and Dickinson, San José, CA, USA). Splenocytes (2 × 10⁵) were then cultured in complete RPMI 1640 media (GIBCO, Thermofisher, Waltham, MA, USA) containing 10% fetal bovine serum, in the absence or presence of MOG_35–55 peptide (Espikem, Prato, Italy) (10 µg/mL). Supernatants were collected after 72 h of culture and kept at −20 °C until use. IFN-γ, IL-17A and IL-10 cytokines were quantified by ELISA MAX™ Standard Set Mouse according to the manufacturer’s instructions (Biolegend, San Diego, CA, USA).