Comprehensive Analysis of ZIKV-Specific Immune Responses

Flow cytometry analysis was performed to detect ZIKV-specific cellular immune responses as previously described [29 (link)]. Briefly, splenocytes (2 × 10⁶ cells/well) were isolated from immunized mice 3 days post-ZIKV challenge, and treated with 1 × Red Blood Cell Lysis Buffer (Biolegend, San Diego, CA, USA), followed by incubation with a ZIKV EDIII peptide mixture (final concentration of 5 μg/mL) (Table 1) in the presence of brefeldin A (5 μg/mL, Biolegend), and cultured at 37 °C for 12 h. After stimulation, the cells were washed with PBS, and stained for surface markers using PerCP/Cy5.5 anti-mouse CD8-positive (CD8⁺), FITC-anti-mouse CD4-positive (CD4⁺), and AF700 anti-mouse CD45 antibodies (Biolegend). After fixation and permeabilization, the cells were further stained for intracellular markers using PE anti-mouse interferon-gamma (IFN-γ), Brilliant Violet 711^TM anti-mouse interleukin 4 (IL-4), and Brilliant Violet 605^TM anti-mouse IL-17 antibodies (Biolegend), which were analyzed using a flow cytometer (BD LSRFortessa 4 system).