To study the chemotactic potency of CXCL2, CXCL8, and CXCL5 forms for CD14+ human monocytes, 48-well Boyden chamber cell migration assays were performed (43). Serial dilutions of CXCL5(1–78), [Cit9]CXCL5(1–78), CXCL5(9–78), and [Cit9]CXCL5(9–78) were prepared in HBSS buffer enriched with 0.5% (v/v) human serum albumin (HSA). Monocytes were suspended in the same buffer at a final concentration of 2 × 106 cells/ml. The lower compartment of the Boyden chamber was filled with chemokine dilutions (30 μl per well) and was covered with a 5-μm pore size polyvinylpyrrolidone-treated polycarbonate membrane (GE Water & Process Technologies). Cells were added to the upper part of the chamber (50 μl per well). After incubation for 2 hours at 37°C, membranes were fixed and stained (Hemacolor Solution I–III, Merck). The numbers of migrated cells were microscopically counted in 10 separate fields for each test condition. CIs were calculated through dividing the number of monocytes that migrated in response to chemokine dilution by the number of cells that migrated in response to buffer alone.

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