Il 2 elisa kit

To detect antigen presentation to T-cells, splenic B-cells from F1 mice of a crossing between B10.BR-H2^k2 H2-T18^a/SgSnJJrep and MD4 mice were co-cultured with 3A9 T-cell hybridoma cells (ATCC CRL-3293) at equal concentrations (3.75 × 10⁶ cells/ml). Cells were incubated in DMEM supplemented with 5 % FBS and 0.05 mM 2-mercaptoethanol for 72 hr in the presence or not of soluble HEL or DEL-I (10 µg/ml), or of beads coated with HEL, DEL-I or Tf (1:4 cell: bead ratio). After incubation, the concentration of IL-2 in the supernatant was measured using an IL-2 ELISA kit (Biolegend).

The splenocytes were separated from immunized mice on day 7 or day 94 after the final immunization and resuspended in complete RPMI-1640 supplemented with 10% FBS, 100 mg/ml streptomycin, and 100 units/ml of penicillin. For in vitro stimulation, a total of 1 × 10⁶ splenocytes were incubated with RBD9.1 or RBD (10 μmol) in 200 μl of complete RPMI-1640 for 48 h at 37°C in a humidified atmosphere containing 5% CO₂. Splenocytes stimulated with Phorbol 12-myristate13-acetate/ionomycin (PMA/I) (Sigma-Aldrich) served as positive controls. The cytokines IFN-γ and IL-2 in media or serum were detected by IFN-γ ELISA kit (BioLegend) and IL-2 ELISA kit (BioLegend), respectively, according to the manufacturer’s instructions.