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. 2013 Mar 14;8(3):e58744. doi: 10.1371/journal.pone.0058744

Figure 2. Real-time chemotaxis of different murine macrophage populations to murine CCL3.

Figure 2

Panels A, D, G & J are representative FACS plots of the four different murine macrophage populations tested in the RTCA-DP system chemotaxis assay. (A) Injection with 2% Bio-Gel (polyacrylamide beads) typically yields 60–70% monocyte/macrophages which are collected after 4 days following the initial injection (assessed by staining with F4/80, 7/4 & CD11b antibodies). (B) 4×105 Bio-Gel elicited peritoneal macrophages were allowed to migrate toward indicated concentrations of murine CCL3 for 3 h. (C) Migration was measured for each curve of each concentration using slope (set between 15–60 mins) analysis. (D) Injection with 4% Thioglycollate generates 70% F4/80+ 7/4− macrophages which were harvested 4 days post injection. (E) 4×105 Thioglycollate-elicited peritoneal macrophages were allowed to migrate toward indicated concentrations of murine CCL3 for 3 h (G). (F) Migration was measured for each curve of each concentration using slope analysis. (G) Lavage of the peritoneal cavity in non-elicited conditions yields ∼30% F4/80hi resident peritoneal macrophages. (H) 4×105 Resident peritoneal macrophages were allowed to migrate toward indicated concentrations of murine CCL3 for 3 h. (I) Migration was measured for each curve of each concentration using slope analysis. (J) Bone marrow cells cultured over 7 days in the presence of L929 conditioned media +10% FBS generated a 92% pure population of macrophages which expressed both CD11b and F4/80. (K) 0.5×105 BMDM were allowed to migrate toward indicated concentrations of murine CCL3 for 3 h. (L) Migration was measured for each curve of each concentration using slope analysis. Data are shown as migration index (i.e. fold change over media alone) for slope analysis. Data are the mean ± SEM of 3–7 independent experiments, each concentration with 2–3 technical replicates per experiment. Statistical analysis was performed by one-way ANOVA and Dunnett's multiple comparison post-test. *, p<0.05, **, p<0.01, ***, p<0.01 relative to media alone.