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. 2013 Sep 23;191(9):4769–4777. doi: 10.4049/jimmunol.1301653

FIGURE 2.

FIGURE 2.

Functional characterization of anti-porcine CD14 Ab rMil2. (A) Whole porcine blood was incubated with 150 μg/ml FITC-conjugated Mil2 (FITC-Mil2) and increasing concentrations of unlabeled rMil2 (○), control IgG2/4 (eculizumab) (□), the original clone Mil2 (●) or mIgG2b isotype control (▪). Remaining FITC-Mil2 binding to the granulocyte population was recorded as median fluorescence intensity of using flow cytometry. Fluorescence intensity in presence of FITC-Mil2 alone was set to 100%. (B and C) Release of the proinflammatory cytokines IL-1β (B) and TNF (C) from porcine whole blood induced with 1 × 105/ml E. coli strain LE392 in presence of increasing concentrations of rMil2 (○), Mil2 (●), control IgG2/4 (□), or mIgG2b (▪). After 120 min incubation, plasma cytokine levels were determined with ELISA, and those measured in absence of any exogenous Ab were set to 100%. (D) Porcine whole blood was incubated with increasing concentrations of rMil2 or Mil2, or ctrl IgG2/4 or mIgG2b isotype controls (up to 71.4 μg/ml). Platelet counts were determined by flow cytometry in routine analysis. Significance for differences in values for rMil2 (○) and Mil2 (●) was calculated by two-way ANOVA and Bonferroni post test (***p < 0.001). (E) Blood slides from samples containing 71.4 μg/ml rMil2 or Mil2 were stained with nuclear stain and investigated by light microscopy. Pictures were taken at a 1000-fold magnification. WBCs, RBCs, and platelets (thrombocytes [TRCs) are indicated by arrows. The single asterisk in the picture for Mil2 indicates a platelet aggregate with surrounding leukocytes that was not observed in rMil2-treated blood. (F) Porcine whole blood was incubated with 50 μg/ml rMil2 (○), Mil2 (●), control IgG2/4 (□) or mIgG2b (▪). After 120 min, plasma levels of IL-8 were measured using ELISA. Data are given as mean and SEM for (A, D, and F) (all n = 3) and as mean and range for (B) and (C) (n = 2). Significance for difference in values for Mil2 and rMil2 (***p < 0.001) was calculated by one-way ANOVA and Tukey post tests. n.s., not significant.