CCL4-mediated inhibition of CXCL12-induced chemotaxis of bone marrow B cells: specificity controls for CCL4.

To assess whether the inhibition of chemotaxis to CXCL12 was specific for CCL4, we used the following controls: (1) cells preincubated at 37°C with 1000 ng/mL CCL4 for 30 minutes, then added into upper well of the chemotaxis chamber (third bar); (2) 1000 ng/mL heat-denatured CCL4 (fourth bar); and (3) 1000 ng/mL CCL4 preincubated for 30 minutes with CCL4-neutralizing antibody (fifth bar). The degree of inhibition is expressed as the percent migration of CCL4-treated cells toward CXCL12 (second bar), compared with the migration of cells pretreated with medium alone (first bar). Results acquired for the pre–B-cell subset are presented. Similar results were obtained for the other bone marrow B-cell subsets (pro-B, immature B, mature B cells). The means (± SD) of 2 experiments, each done in triplicate, are presented. Migration toward CXCL12 of untreated cells was considered 100%. *indicates the percent migration of CCL4-treated cells is statistically significant (P < .005, pairedt test 2-sided) compared with the migration of untreated (medium only) cells toward CXCL12. In contrast, the percent migration of cells treated with CCL4 controls was not statistically different.