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. 2018 Jun 5;9:1195. doi: 10.3389/fmicb.2018.01195

FIGURE 6.

FIGURE 6

Tetra-antigen TMV vaccine induces potent recall response in immunized mice. (A) C57BL/6 mice (n = 3–4) were immunized as shown. Mice were sacrificed on day 84 post-primary immunization and single cell spleen suspensions were prepared. BMDMs isolated from syngeneic C57BL/6 mice were infected either with F. tularensis LVS (B) or F. tularensis SchuS4 (C) at an MOI of 100 (100:1 bacteria:cell ratio), for 2 h followed by gentamicin treatment for 2 h. The infected BMDMs were then overlaid with splenocytes isolated from immunized mice at 1:1 ratio. Splenocytes isolated from naïve mice (n = 3) were used as controls. The culture supernatants from co-culture were analyzed for IFN-γ and IL-17 levels 24, 48, and 120 h later by CBA. The limits of detection for IL-17 and IFN-γ by CBA are 18.9 and 3.7 pg/ml, respectively. The results are representative of two independent experiments. The data were analyzed by one-way ANOVA followed by Bonferroni’s correction and P-values were determined.