Macrophage chemotaxis assay

In vitro macrophage chemotaxis assays were performed in 96-Transwell plates (Corning). Approximately 2 × 10⁵ PMA-differentiated THP-1 macrophages or M-CSF mBMDMs were plated in the upper chambers while the lower chambers were filled with the CM. After 6 h of incubation, the medium was aspirated from upper and lower chambers. Chambers were washed once with PBS followed by incubation with calcein-AM at 37°C for 1 h with gentle tapping every 15 min. After removal of the filter, the plates were read with an Epoch microplate reader (485 nm for excitation and 520 nm for emission). Chemotactic effects were presented as chemotaxis index, a ratio of quantitative fluorescence of cells incubated with the CM to cells incubated with serum-free DMEM.