Measurement of in vitro cytokine production and proliferation

Single cell suspensions from spleens were prepared and 5x10⁶ cells were cultured in vitro with OVA (0.2 mg/ml) in culture medium (RPMI medium supplemented with 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin). Cytokines (IL-5, IL-10, IL-13, IFN-γ) were measured in supernatants of spleen cells at day 3 of restimulation with OVA by commercially available ELISA (R&D Systems, USA), according to the manufacturer’s instructions. To determine proliferation, OVA-restimulated splenocytes were analyzed using a cell titre glow assay (Promega, Germany) according to the manufacturer’s instructions. In detail, 0.1 ml cell suspension and 0.1 ml freshly prepared glow reagent were mixed and measured using a GloMax Multi-Detection System (Promega, Germany).