Segments of mice spleens stored in RPMI 1640 were gently squeezed and passed through a sterile filter (250 mm, 12–13 cm, AlterChem Co, Athens, Greece) for the collection of splenocytes. After three serial washings, cells were counted on Neubauer plates with trypan blue for exclusion of dead cells. A total of 5 x 106 cells/ml were then incubated into sterile 24-well plates in RPMI-1640, supplemented with 2 mM glutamine, 10% fetal bovine serum, 100 U/ml of penicillin G and 0.1 mg/ml of streptomycin in the absence or presence of 10 ng/ml LPS of E. coli O55:B5 or 5 x 105 cfu/ml heat killed Candida αlbicans. After 24 hours or 5 days of incubation at 37°C in 5% CO2, the plates were centrifuged, and the supernatants were collected. Concentrations of TNFα was measured in duplicate in supernatants from stimulated splenocytes, in tissue supernatants and in serum via enzyme- linked immunosorbent assays (ThermoFisher Scientific, California, USA). The lower detection limit was 39 pg/ml. Additionally, IL-1α, IL-1β, IL-6, IL-10, IL-12, IL-27, IFNγ and IFNβ in serum were determined by the LEGENDplex mouse TH 1/2 cytokine panel (13-plex) (Biolegend, California, USA) according to the manufacturer’s instructions.

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