Anti-CXCR4 antibody reduces CXCR4 surface expression, and blocks migration toward CXCL12 and BMSCs.

<p>(A) Relative MFIR of CXCR4 surface staining after incubation of Raji transfectants with 100 µg/mL of anti-CXCR4 antibody for 30 minutes. (B) Flow cytometry histograms showing the reduction in CXCR4 expression after incubation with neutralizing anti-CXCR4 antibody. White histograms represent negative unstained controls. (C) CXCR4-blocked cells were stimulated with 100 ng/mL of CXCL12 for 5 minutes and activation of Akt and ERK1/2 proteins analyzed by western blotting. (D) CXCR4-blocked cells were subjected to migration assay for 4 hours at 37°C in 5% CO₂ toward 100 ng/mL of CXCL12 (left panel) or the stromal MS-5 cell line (right panel). Cells in the lower chamber were counted by flow cytometry under a defined flow rate for 5 minutes. Number of migrated cells treated with isotypic control was considered 100%. Results are shown as the mean ± SEM of 4 independent experiments. (E) Cell viability was measured by Annexin V-PI staining in cells incubated with 100 µg/mL of anti-CXCR4 or isotypic control for 48 hours. Results are shown as the mean ± SEM of 4 independent experiments