Role of S100A9 in Arthritis Figure 6. Neutrophil migration across HUVECs in response to S100A9. A) Increasing concentrations of S100A9 were added in the upper wells with neutrophils. IL-8 (5 ng/ml) or buffer was added to the lower wells and neutrophils were allowed to migrate for 2 h at 37uC. Data are the mean 6 SEM of 3 experiments using neutrophils from different donors. *p,0.05, **p,0.01, one-way ANOVA, Dunnett’s multiple comparison test. B) S100A9 prolongs the time for neutrophil migration across endothelial cells. S100A9 (40 mg/ml) was added to the upper well with neutrophils. IL-8 (5 ng/ml) or buffer was added to the lower well and every 30 min for up to 2 h the upper wells were moved to new lower wells. The number of transmigrated cells was determined as described in Materials and Methods. Data shown represent the mean 6 SEM of at least 3 experiments using neutrophils from different donors. doi:10.1371/journal.pone.0045478.g006 PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e45478