Confirmation of the inhibitory effects of netrin-1 on macrophage chemotaxis using a real-time microscopy based migration assay.

<p>BMDMs were incubated with either vehicle, 3 nM or 30 nM netrin-1 for 45 min at 37°C, 5% CO₂ prior to being placed into the upper chamber of an IncuCyte® ClearView chemotaxis plate (5000 cells/well), which had been coated with Matrigel (50 μg/ml for 30 min at 37°C and 30 min at RT). Cells were then allowed to migrate towards vehicle or 100 nM C5a and chemotaxis was measured by imaging the underside of the well every 2 hours and calculating total underside cell area. Representative images of cells migrating towards (<b>A</b>) vehicle or (<b>B</b>) 100 nM C5a and cells migrating towards 100 nM C5a that had been treated with (<b>C</b>) 3 nM or (<b>D</b>) 30 nM netrin-1. The pores connecting the upper and lower chambers are masked in yellow and cells that have migrated to the underside of the well are masked in pink. (<b>E</b>) A representative real-time trace of total cell area on the underside of the well against time. (<b>F</b>) Quantification of BMDM chemotaxis by maximum cell area reached on the underside of the well. Data are mean + SEM, n = 4 biological replicates with 4 technical replicates per condition. Statistical analysis was conducted by one-way ANOVA with Dunnett’s multiple comparisons correction.* P < 0.05 for indicated comparisons.