Lymph node and spleen CD4+ and CD8+ T cells from immunized mice (collected at day 28) and those from PBMCs (collected at day 14 and day 28) were evaluated for antigen-specific responses. PBMCs were isolated by sucrose gradient density separation method using Histopaque (Sigma-Aldrich) as described earlier (65). Briefly, isolated PBMCs, lymph node cells, and splenocytes were cultured for restimulation with DENVrEDIII protein (10 μg ml−1) in the presence of GolgiStop and GolgiPlug (BD Biosciences) at 37°C for 10 hours. The restimulated cells were stained with Alexa Fluor 488–labeled anti-mouse CD8 (BD Biosciences) and PerCP (eBioscience) or fluorescein isothiocyanate–labeled anti-mouse CD4 antibodies (BD Biosciences) for 60 min at 4°C. The cells were further washed three times with FACS buffer, fixed with 4% paraformaldehyde, permeabilized using 1X Perm Wash Buffer (BioLegend), and stained using APC-labeled anti-mouse IFN-γ (BD Biosciences) and phycoerythrin-conjugated anti-mouse IL-2 antibody (BD Biosciences), diluted in 1X Perm Wash Buffer for 60 min at room temperature. Cells were washed with FACS buffer, and data were acquired on a BD LSRFortessa (BD Biosciences) cytometer and analyzed using FlowJo (Tree Star Inc.).

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