Fitc anti mouse cd4 or percp cy5.5 anti mouse cd8 antibody

Flow cytometry analysis was performed to evaluate CD4⁺ and CD8⁺ T cell depletion and ZIKV-specific CD8⁺ T cell responses in the challenged mice (Guerrero et al., 1986 (link); Zhang et al., 2016 (link)). For analysis of CD4⁺ and CD8⁺ depletion in whole blood and splenocytes, peripheral blood cells and splenocytes were treated with 1 × Red Blood Cell Lysis Buffer (Biolegend), and stained with FITC-anti-mouse CD4 or PerCP-Cy5.5-anti-mouse CD8 antibody (BD Biosciences), followed by flow cytometry analysis using BD LSRFortessa 4 system. For analysis of ZIKV-specific CD8⁺ T cell responses, the above-treated splenocytes (2 × 10⁶ cells/well) were incubated with ZIKV NS3 overlapping peptides (0.25 nM/peptide, final concentration 5 μg/ml) in the presence of 5 μg/ml brefeldin A (Biolegend), and cultured at 37°C for 5 h. After stimulation, the cells were washed with PBS and stained for surface marker using PerCP/Cy5.5 anti-mouse CD8a. After fixation and permeabilization, the cells were stained for intracellular markers using FITC-anti-mouse IL-2, PE-anti-mouse IFN-γ, and Brilliant Violet 421-anti-mouse TNF-α antibodies (BD Biosciences), followed by analysis using flow cytometry as described above.