Isotype distribution of GC B cells after single immunization with polyanhydride nanovaccine. BALB/c mice were injected s.c. with 500 μg total of gp41-loaded 20:80 CPTEG:CPH nanovaccine. dLNs were harvested 12 days after the challenge. Cell suspensions were stained with PNA, anti-B220 mAb, anti-IgM mAb and either goat anti-IgG1, IgG2a+IgG2b (BALB/c), or IgG3 specific Abs. Stained cells were analyzed by flow cytometry. Panel A shows the gating strategy used in flow cytometry for identification of IgM+, IgG1+, IgG2a+/IgG2b+, and IgG3+ germinal center B cells from lymph nodes of BALB/c mice. IgM+, IgG1+, IgG2+ and IgG3+ B cells are shown as a percent of total GC B cells in Panel B. Each bar represents mean±SEM. n =4 mice at each time point.