CD9 does not affect CXCR4 localization and internalization. Migration assay. Chemotactic migration toward CXCL12 (100 ng/mL) of REH cells after preincubation with 1 μg/mL anti-CD9 antibody and 20 µM plerixafor (A) or CCX771 (100 nM) (B) was measured in a Boyden chamber. The migration rates are represented using a scatter dot plot with the histograms indicating the means of 6 independent experiments. *P < .05 in Wilcoxon test. (C) Immunofluorescence. Cells were subjected to cytospin centrifugation and fixed in 4% paraformaldehyde. The CXCR4, CXCR7, and CD9 proteins were labeled with mouse anti-CD9 (1:50), rabbit anti-CXCR4 (1:50), and rabbit anti-CXCR7 (1:500) antibodies. Confocal imaging of serial Z stacks was performed with a Leica SP5 confocal microscope equipped with a 63×/1.4 oil-immersion objective and ×6 zoom. The yellow spots on the merged images indicate the colocalization of CXCR4 and CD9. The images are representative of 3 independent experiments. (D) REH cells were stimulated with CXCL12 (200 ng/mL) for 20 minutes at 37°C. CXCR4 expression on the membrane was assessed by flow cytometry. The histograms show CXCR4 expression in the absence of stimulation (black peaks) and after stimulation (gray peaks), as assessed by flow cytometry. The black lines indicate the fluorescence of REH cells labeled with an isotypic control antibody.