Spleen lymphocytes were seeded into anti-mouse IFN-γ mAb pre-coated 96-well plates at 2 × 105 cells (100 μL/well) and stimulated with 10 μg/mL purified protein for overnight incubation; phorbol 12-myristate 13-acetate (PMA) was used as a positive control. The ELISPOT assay was performed using a mouse IFN-γ ELISPOT Kit (Dakewe) according to the manufacturer's instructions (Ranieri et al., 2014 ). Spots were counted using an immunospot reader (Cellular Technology Limited, USA).
Spleen lymphocytes (2 × 106 cells) were firstly stimulated for 2 h with 10 μg/mL of purified E1-DIII + NS1-2 and E4-DIII + NS1-3 protein as a specific antigen. Then the treated cells were continue cultured in the presence of 10 mg/mL monensin and brefeldin A in complete RPMI 1640 (Gibco) overnight. Stimulated cells were first incubated and stained with Fixable Dye eFluor 506 and the CD8-Percp-eFluor 710, CD3e-eFluor 450 (eBioscience), CD45-APC-Cy7, and CD4-FITC (BD Biosciences) surface markers. Then, cells were fixed in permeabilization buffer (Thermo Fisher Scientific) and stained with IFN-γ-PE-eFlour 610, IL-4-PE-Cyanine7, and TNF-α-APC (eBiosciences). All labeled lymphocytes were analyzed on a FACSAria III flow cytometer (BD Biosciences), and the data were analyzed using FlowJo V10.
Spleen lymphocytes (2 × 106 cells) were firstly stimulated for 2 h with 10 μg/mL of purified E1-DIII + NS1-2 and E4-DIII + NS1-3 protein as a specific antigen. Then the treated cells were continue cultured in the presence of 10 mg/mL monensin and brefeldin A in complete RPMI 1640 (Gibco) overnight. Stimulated cells were first incubated and stained with Fixable Dye eFluor 506 and the CD8-Percp-eFluor 710, CD3e-eFluor 450 (eBioscience), CD45-APC-Cy7, and CD4-FITC (BD Biosciences) surface markers. Then, cells were fixed in permeabilization buffer (Thermo Fisher Scientific) and stained with IFN-γ-PE-eFlour 610, IL-4-PE-Cyanine7, and TNF-α-APC (eBiosciences). All labeled lymphocytes were analyzed on a FACSAria III flow cytometer (BD Biosciences), and the data were analyzed using FlowJo V10.