Aec substrate solution

The membranes of the Millipore plates (Millipore; MSIPS4510) were activated with 35% ETOH prior to coating. The membranes were coated with 10 μg/ml of each of the following reagents diluted in DPBS: NP4-BSA; NP14-BSA, Imject BSA, Imject KLH. The plates were incubated at 4°C overnight. Plates were washed and blocked with 10% goat serum (Gibco) diluted in tissue culture medium (RPMI 1640/Penicillin-Streptomycin/L-Glutamine and 5x10⁻⁵ M 2-mercaptoethanol) for 2 h at room temperature. Mouse splenocytes were resuspended in tissue culture medium supplemented with 10% FBS, added at 2×10⁶ cells per well and cultured for 48 h at 37°C. The plates were developed using AEC substrate solution (BD; 551951) and sent for analysis to Cellular Technology, LTD.

The ELISPOT 96-well plates (BD, San Jose, CA, USA) were coated with 100 μL of anti-mouse IFN-γ (5 µg/mL in coating buffer) at 4 °C overnight. The following day, plates were washed and blocked with blocking buffer for 2 h at 37 °C. Then, 100 μL freshly isolated splenocytes (5 × 10⁵ cells) from the immunized mice were added to each well and stimulated with avian influenza H7N9 viruses A/Wuxi/1/2013 (H7N9) (GenBank: KF034914.1) at 37 °C for 40 h with 5% CO₂. After that, cells were washed, biotinylated anti-mouse IFN-γ was added to each well and incubated for 2 h at room temperature. Then plates were washed and incubated for 1 h at room temperature with streptavidin-HRP. Finally, AEC substrate solution (BD) was added and spots were counted by ImmunoSpot Analyzer (Cellular Technology Ltd., Cleveland, OH, USA).