Collagen from rat tail

Chemotaxis assays were performed using 8 μm pore size cell culture inserts within 24-well plates (Corning, USA) as previously described [22 (link)]. FTC133 or WRO82-1 in exponential phase were cultured with FBS free medium (plus 0.2 % BSA, Genebase Gene-Tech, China) for 24 h and then the medium was harvested as condition medium (CM). THP-1 cells (5 × 10⁵/ml) were seeded into the inserts pre-coated with collagen from rat tail (Sigma, USA), and CM/ mock medium /CCL15 neutralizing antibody was added in the lower well. After 12 h of incubation, cells in the upper membrane surface were removed with cotton swabs. Cells on the lower membrane surface were fixed with methyl alcohol and stained with 0.1 % crystal violet (Sigma, USA). The cells of five randomly selected fields per well were counted using the Axiovert 25 microscope (Carl Zeiss, Germany) under 400 magnifications. Three replicate measurements were included in a single experiment.

The DMR assay was performed on stable HEK293-Glo-MOR cells as described^{73 (link)}. Cells were seeded (30 µL per well of a 300,000 cells/mL suspension) onto a Cellular Label-free 384-well microplate (Perkin Elmer) previously coated with collagen from rat tail (Sigma Aldrich) and containing 10 µL medium per well. Plate was let in a hood at room temperature for 30 min before overnight incubation à 37 °C in a humidified CO₂ incubator. The day of the assay, four careful washes of the cell layer with Hepes buffer (see “cAMP accumulation” description) were done, after which the plate was let to equilibrate with 30 µL per well Hepes buffer for 2 h in a EnSpire 2300 Multimode Plate Reader (Perkin Elmer). DMR was then monitored in the apparatus at room temperature before and after compound addition (10 µL added per well). Three independent experiments were done in duplicate or triplicates. For results representation, kinetic curves of control conditions (buffer-treated cells) were subtracted.