Demonstration of chemotaxis and its inhibition by Pertussis toxin (PTX) and cytochalasin D.

<p>To test that the assay was detecting true macrophage chemotaxis, rather than chemokinesis or fugetaxis, the following three conditions were tested; CCL5 added to the bottom well alone (<i>test chemotaxis</i>), equal concentrations (5 nM) of murine CCL5 added to both top and bottom wells (<i>test chemokinesis</i>) or CCL5 added to the top well alone (<i>test fugetaxis</i>) (<b>A</b>) Single trace of a single experiment with four technical replicates shows Bio-Gel elicited macrophages migrate towards murine CCL5 (green), but display no chemokinesis (red) or fugetaxis (blue). Control (pink) had chemotaxis buffer alone in the bottom well. Bio-Gel elicited macrophages were pre-incubated for 90 mins at 37°C with PTX (200 ng/ml). (<b>B</b>) A representative trace shows that pre-treatment of Bio-gel elicited macrophages with PTX significantly inhibits chemotaxis toward both murine CCL2 and CCL5. Data are shown as a percentage of 10 nM CCL2 or CCL5 induced migration for (<b>C</b>) Slope (<b>D</b>) Max-min CI and (<b>E</b>) AUC. Data are the mean ± SEM of three independent experiments with 3–4 technical replicates per experiment. Vehicle alone was omitted in these sets of experiments in order to have all five conditions on a single CIM-16 plate. (<b>F</b>) Bio-Gel elicited macrophages were pre-incubated for 90 mins with either 200 nM cytochalasin D, an inhibitor of F-actin polymerisation. A representative trace is shown (<b>F</b>). Data are shown as a percentage of 5 nM CCL2 induced migration for (<b>G</b>) Slope (<b>H</b>) Max-min CI and (<b>I</b>) AUC. Data are the mean ± SEM of four independent experiments with 3–4 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 CCL2 or CCL5 alone.