Figure 4

PMN migration by the supernatant of early and mid CL culture and PMN migration levels with or without anti-IL-8 Ab pre-treatment. Before PMNs migration experiment, we evaluated the purity of PMNs separation from white blood cells using flow cytometry. Figure 4A indicates the plotting data of peripheral white blood cells (not separate between PMNs and pheripheral blood mononuclear cells (PBMCs)). After removing PBMCs layer using Lymphoprep, we observed clear separation of PMN population (Figure 4B) and the purity of PMNs was > 95% and these cells resulted in nearly pure granulocyte populations as determined by flow cytometric evaluation. PMN migration by the supernatant obtained from early- and mid-phase CL cultures are shown in Figure 4C. The supernatant of the early CL significantly induced PMN migration as well as IL-8 (50 ng/ml as a positive control) compared with control. Figure 4D indicates the reduction ratio of migrated PMNs using the supernatant of the early CL with anti-IL-8 monoclonal antibody or control IgG. All values are the mean ± SEM. Different superscript letters indicate significant differences (P < 0.05) as determined by ANOVA followed by the Bonferroni's multiple comparison test.