FIGURE 3. Optimization of staining conditions to identify memory B cells that bind AF-DENV.

Memory B cells were identified as live CD3⁻CD14⁻CD19⁺CD27⁺IgM^– cells, as delineated in the gating strategy outlined in Supplementary Figure 1. (A) Different concentrations of AF-DENV were added to DENV immune PBMC at 4°C for 30 min. Frequencies represent AF-DENV⁺ cells within the CD19⁺CD27⁺IgM⁻ B population. (B) Flow plots depict frequencies of AF-DENV⁺ memory B cells when binding was performed at 4°C or 37°C. (C) Representative flow plots of PBMC from immune and naïve donors showing frequencies of AF-DENV⁺ (i and ii) and AF-VERO⁺ (iii and iv) memory B cells. (D) Representative flow plots depicting the use of AF-VERO preparations to delineate non-specific staining of PBMC from immune and naïve donors. AF647-VERO⁺AF488-DENV⁺ memory B cells were excluded from the gating strategy to identify DENV⁺ B cells. Numbers within the gates in the dot plots represent the frequencies of AF488-DENV⁺ AF647-VERO⁻CD19⁺CD27⁺IgM⁻ B cells in immune and naïve donors. (E) Numbers within the gates in the dot plots represent the frequencies of AF488-DENV-1⁺AF647-DENV1⁺ CD19⁺CD27⁺IgM⁻ B cells in an immune and naïve donor.