Chemotaxis Assay of Monocyte Migration

Conditioned or control media were diluted 1:2 in chemotaxis medium (PBS with 1% albumin, low endotoxin; Sigma). Monocytes were freshly isolated from HDs after obtaining written informed consent using negative MACS depletion as described previously^{20 (link)} and resuspended in chemotaxis medium. The diluted media were added in the lower chambers of a 5 μm chemotaxis assay plate (96 well ChemoTX®, NeuroProbe, Gaithersburg, MA, USA) and 100,000 monocytes were transferred to the upper chamber. After 2 h, chemotaxis was quantified by measuring the DAPI (4,6 diamidino-2-phenylindole) signal of migrated monocytes as described previously.^{20 (link)}When measuring inhibitor effects, both media and monocytes were incubated for 30 minutes (min) on ice with the indicated inhibitors directly prior to the migration assay. The following chemokine receptor inhibitors were used: 1 μg/mL CCR1 inhibitor BX471 (Sigma), 1 μg/mL CCR2 inhibitor INCB3284 (Tocris Bioscience, Bristol, UK), 1μM CCR3 inhibitor SB328437 (Tocris), 1 μM CCR5 inhibitor Maraviroc (Apexbio, Houston, TX, USA), 1μM CXCR4/7 inhibitor Plerixafor (Apexbio), and 0.1 μg/mL IL-10 neutralizing antibody (R&D Systems, Minneapolis, MN, USA).