Trans-well migration assays to ADP was performed as previously described (De Simone et al., 2010; Moore et al., 2015). iMGLs (5.5×104 cells/well) were cultured in serum-free basal media without cytokines pre-exposed to DMSO or PSB0739 (50 μM, Tocris) for 1hr at 37ºC in 5% CO2 cell culture incubator. Cells were then washed three times with basal medium and plated in trans-well migration chambers (5 μm polycarbonate inserts in 24 wells; Corning) containing Adenosine 5′-phosphate (ADP, 100 μM; Sigma) in the bottom chamber in 37ºC in 5% CO2. After 4 hours, cells were washed three times with PBS (1x) and fixed in PFA (4%) for 15 minutes at room temperature. Cells were stained with Hoechst stain for 10 mins to visualize nuclei of cells. A blinded observer counted total cells per slide and then scrubbed cells off top surface using a cotton-swab, washed with PBS, and recounted to record migrated cells. Migration was reported as migrated over total cells per well. Fluorescent images of cells were captured using Olympus IX71 inverted microscope.
