Anti mouse il 2 clone jes6 1a12

Splenocytes (1 × 10⁶ cells/ml, 0.8 ml/well) were seeded in 48-well flat bottom plates and treated with CBD (0.5 - 10 μM) or VH (0.1% ethanol) for 30 min. Cells were stimulated with S/o, Us/o or CD3/28 stimulation for 1-5 days. Cytokine production in the culture supernatant was assessed by ELISA. Wells were coated overnight at 4°C with 1.0 μg/ml anti-mouse IL-2 (clone JES6-1A12, BioLegend) in coating buffer (0.1 M carbonate-bicarbonate buffer, pH 9.6). Wells were washed three times each with 0.05% Tween-20 in PBS (PBST, pH 7.4) and deionized water (DW), then blocked with 3% BSA in PBS for 1 h at RT. After washing the wells, samples were added to each well and incubated for 1 h at RT. Again after washing, wells were incubated with 1.0 μg/ml biotin-conjugated anti-mouse IL-2 (clone JES6-5H4, BioLegend) for 1 h at RT. Wells were washed and treated with 100 μl of Avidin-conjugated with horseradish peroxidase (HRP-Avidin; 1:500, BioLegend) for 1 h at RT in the dark. For color development, wells were washed then 100 μl of 3,3′,5,5 -tetramethylbenzidine (TMB) substrate set (BioLegend) was added for 30 min followed by the addition of 100 μl of 2 N sulfuric acid (H₂SO₄) to terminate the reaction. Optical density (OD) was measured at 450 nm within 30 min of reaction termination. Quantification of cytokine was performed by generation of a standard curve.

5×10⁴ enriched CD4⁺ T cell from WT^KL, C4^KL, or WT^CL mice were cocultured with 1X10⁵ transduced I-A^b+ M12 cells in triplicate in a 96 well round bottom plate in RPMI with 5% FBS (Omega Scientific), Pen-Strep+L-Glutamine (Cytiva), 10ng/ml ciprofloxacin (Sigma), and 50mM beta-2-mercaptoethanol (Fisher) in the presence of titrating amounts of OVA 323–339 peptide starting at 10µM OVA and a 1:3 titration. The supernatants were collected and assayed for IL-2 concentration by ELISA after 16 hours of co-culture at 37ºC. Anti-mouse IL-2 (clone JES6–1A12, BioLegend) antibody was used to capture IL-2 from the supernatants, and biotin anti-mouse IL-2 (clone JES6–5H4, BioLegend) antibody was used as the secondary antibody. Streptavidin-HRP (BioLegend) and TMB substrate (BioLegend) were also used.