A half million cells were resuspended into 100 μl of RPMI 1640 medium and then added to the upper chamber of a 24-well Transwell plate (Corning, Corning, NY). The lower chamber was filled with 600 μl of medium premixed with CXCL12 (40 ng/ml). The plate was incubated at 37°C for 2 hours, and then the upper chamber was removed and cells in the lower chamber were counted. To ensure accurate enumeration of cells, only Z2 Coulter Particle Count and Size Analyzer (Beckman Coulter) was used. Where indicated, different concentrations of R10015 (26) or DMSO were added to cell culture and incubated for 1 hour at 37°C before adding cells to the upper chamber. Cells were also treated with the anti–human α4β7 integrin antibody (Act-1) or the control mouse immunoglobulin G1 (IgG1) antibody for 15 min before adding cells to the upper chamber. Act-1 was also added to the lower chamber (1 μg/ml) with CXCL12 (40 ng/ml). Multiple donors were used for the chemotaxis assay.

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