Ova 5

For all in vitro stimulation assays, 2 × 10⁵ cells were cultured at 200 µl per 96-well of RPMI 1640 complete medium in the presence of either ODN1585 (CpG A, 1 µM; Invivogen), ODN1826 (CpG B, 1 µM; Invivogen), lipopolysaccharide (LPS, 1 µg ml⁻¹; Sigma-Aldrich), low endotoxin ovalbumin (OVA LE; 1 µg ml⁻¹; Invivogen), or grade V ovalbumin (OVA V, 1 µg ml⁻¹; Sigma-Aldrich). Control cells were left untreated. If not stated otherwise, recombinant murine (rm) β-NGF (100 ng ml⁻¹; R&D Systems) was used according to previous studies of a mouse model of OVA-mediated allergic asthma (28 (link)). The production of mouse IFNα was measured 24 h after set-up using an ELISA (eBioscience). Murine IL-6 and TNFα levels were assessed using a CBA (BD Biosciences). The remaining cells were used for flow cytometric analysis.

Spleens were excised and single cell suspensions were obtained and re-stimulated as previously described (85 (link)). Cells were washed and re-suspended in complete medium [RPMI 1640 supplemented with 10% FCS, 1% glutamine, 1% penicillin-streptomycin, 1% Na-pyruvate, 1% non-essential amino acids (Gibco, Life Technologies GmbH) and 50µM 2-β-mercaptoethanol (Sigma-Aldrich)], plated in 96-well at a concentration of 2x10⁵ cells/well and cultured for 72 h with medium alone or with OVA V (5 µg/ml; Sigma-Aldrich). Their supernatants were analyzed for cytokine expression using the V-Plex proinflammatory panel 1 mouse and V-plex IL-9 kit (MesoScaleDiscovery) according to manufacturer’s instructions.