Mouse il 2 ready set go kit

Splenic T cells or purified CD4⁺ T cells (Pan T cell isolation kit, CD4⁺CD25⁺ Regulatory T cell isolation kit, Miltenyi Biotec) were treated with the indicated concentrations of IFNβ (Biogen Idec) for 16 h or as indicated prior to stimulation with either 10 μg/mL anti-CD3 and 2 μg/mL anti-CD28 (eBioscience) or 5 ng/mL phorbol myristate acetate (PMA) and 500 ng/mL Ionomycin (Sigma) for 3 or 6 h and then cells were analyzed for IL-2 protein by intracellular stain or RNA by reverse transcription and quantitative PCR. Mouse IL-2 ready set go kit (eBioscience) was used to measure IL-2 released into the conditioned T cell culture medium. Human PBL's were stimulated with Dynabeads Human T cell activator CD3/CD28 (Invitrogen). Trichostatin A (Sigma) was added to cultures 1 h prior to stimulation with anti-CD3/CD28. For in vivo stimulations OTII TCR transgenic mice were intravenously injected with 100ug Ova_323-336 peptide (Anaspec) 24 hours following injection with 10000 U IFNβ (Biogen Idec). Splenic T cells were removed 4 hours following peptide injection and subjected to intracellular staining for IL-2 and analyzed by flow cytometry.