Recombinant CXCL12 binds to the surface of MS-5 stromal cells previously stripped of endogenous CXCL12 by NOX-A12. (A) MS-5 cells were incubated with 100 nM NOX-A12 or with the inactive variant revNOX-A12 for 1 hour. CXCL12 concentrations in supernatants were quantified by ELISA. Extracellular CXCL12 most likely presented by GAGs was detached from MS-5 cells by NOX-A12. (B) MS-5 cells were washed three times to remove any free NOX-A12 and incubated with 1 nM recombinant human CXCL12 for 3 hours. CXCL12 concentrations in supernatants were quantified by ELISA and the percentage of CXCL12 recovered from supernatants was calculated. The results indicate that recombinant CXCL12 binds to the extracellular binding sites of MS-5 cells that were previously stripped of endogenous CXCL12 by NOX-A12. The unpaired Student t test was used for statistical analysis. Data are representative of 3 independent experiments. *P = .01 to .05; **P = .001 to .01; ***P < .001.