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. 2018 Oct 12;201(10):2879–2884. doi: 10.4049/jimmunol.1800721

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FIGURE 3. — Stimulation of DCs with CD40L beads. (A) BMDCs (unactivated) from B10.A-Rag2^−/− mice were cocultured at 2 × 10⁴ cells per well plus 50 μl of Ni-NTA agarose beads (∼3000 beads/well), which were either coated with ∼15 μg/ml sCD40L (CD40L+beads), or not (beads alone), in the presence or absence of cytokines at 100 ng/ml for 48 h in a 96-well U-bottom plate. CSN was tested for the presence of IL-12 using ELISA. (B) Same conditions as (A), except DCs were preactivated with 200 ng/ml LPS for 22 h, then washed and cocultured with beads alone or beads coated with CD40L. All pairwise comparisons were made using two-way ANOVA with Tukey post hoc tests of two independent experiments of duplicate wells. (C) Same condition as (B); LPS-DCs cocultured with serially diluted CD40L-beads (∼15 μg/ml) in the presence of constant (100 ng/ml) level of IL-4 for 48 h. CSN was tested for the presence of IL-12. Symbols and whiskers represent mean ± SD of triplicates, whereas line represents linear regression. (D) Same as (B); LPS-DCs cocultured with glass-supported Ni-NTA lipid bilayer beads that had been coated with the indicated number of histidine-tagged CD40L molecules in the presence or absence of IL-4 at 100 ng/ml for 48 h. CSN was tested for the presence of IL-12 heterodimer using ELISA and cytokine array. These data are expressed as the mean ± SD triplicate wells, representing two independent experiments. (E and F) Same conditions as (D), except CSN were tested for the presence of TNF-α and IL-6. (G) Same conditions as (A) and (B), except that IL-12 and IL-23 were measured in parallel. The impact of IL-4 on CD40L-induced IL-23 production by LPS-DC (a decrease) was compared with that of IL-4 on CD40L-induced IL-12 production by LPS-DC (an increase). The significance of this difference from a larger decrease in IL-23 to slightly smaller increase in IL-12 was analyzed with three-way ANOVA and a custom contrast to compare a difference of differences between the IL-12 increase and the IL-23 decrease using JMP software of two independent experiments of duplicate wells.