Inf γ

Mesenteric lymph nodes (MLN) cells were isolated from mice and cultured in RPMI culture medium (Lonza) with 100 unit of streptomycin, penicillin (PAA Laboratories) and 10% SVF (Lonza) at 2 × 10⁶ cells/well. Cells were re‐activated with 4 μg ml^–1 pre‐coated anti‐mouse antibody CD3e and CD28 (eBioscience). Concentrations of cytokines IL‐12, IL‐17, IL‐4, TSLP, and INF‐γ (Mabtech) and TGF‐β (R&D), in medium were assessed by ELISA after 48 h of incubation (37°C/5%CO₂).
One centimetre of colonic tissue was weighing and mashed by Gentle MaxTM (Miltenyl Biotec) in 1 ml of PBS plus anti‐protease (Roche). The lysate was centrifuged and the supernatant used to measure cytokine level by ELISA. The cytokines tested were IFNγ, IL12, IL4, IL17, TSLP (Mabtech) and TGF‐β (R&D systems), and the concentration was normalized by microgram of tissue.

Monkey blood was obtained and PBMCs were isolated as previously described.^{21 (link)} Cells were washed twice with phosphate-buffered saline 2% fetal bovine serum (PAA Laboratories, Ontario, Canada) and homogenized at 2 × 10⁶ cells per ml in Roswell Park Memorial Institute 1640 medium (Sigma Aldrich) supplemented with 100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin (Gibco, UK), 2 mm glutamine (Gibco, UK), 5 × 10⁻⁵M 2-mercaptoethanol (Sigma, St Louis, MO, USA) and 5% fetal bovine serum. Finally, 2.5 × 10⁵ cells per well were cultured in 96-well round bottom plates and stimulated with the mock preparation or DENV at multiplicity of infection (MOI) of 0.5.
Concanavalin A (Sigma) was used as a positive control. In all experiments two wells were plated for each antigen. After 4 days of culture, culture supernatants were collected and stored at −80 °C. The culture supernatants were analyzed in duplicate for INFγ concentrations by ELISA using monoclonal antibody pairs (Mabtech INFγ Nacka Strand, Sweden) and the protocol recommended by the manufacturer. The optical density at 492 nm in cells stimulated with mitogen was used to normalize data. Positive response was considered when the IFNγ concentration value in the stimulated PBMCs was twice or higher than the IFNγ concentration in cells without viral stimulus.