Effects of neutralizing anti-CD9 mAb on in vitro homing-related functions of cord blood CD34⁺ cells. (A) Cells were cultured in the presence of an anti-CD9 mAb (ALB6; 10 μg/mL) or an irrelevant isotypic control mAb (IgG; 10 μg/mL), or pretreated with SDF-1 (100 ng/mL) for 4 hours in the presence of the anti-CD9 mAb (ALB + SDF-1) or the isotypic control mAb (IgG + SDF-1). Cell-surface expressions of CD9 and CXCR4 were detected by flow cytometry. Representative dot plots of CD9 and CXCR4 expression and the percentage of cells in each quadrant are shown. Cell migration through (B) bare filters (n = 4) or across (C) HUVEC monolayers (n = 4) to a gradient of SDF-1 (100 ng/mL) was determined using Transwells. The percentage migration was calculated as follows: (number of migrated cells/input cell number) × 100. (D) Actin polymerization assay: cells were stimulated with SDF-1 (100 ng/mL) for the indicated times, and the mean phalloidin-FITC fluo-rescence intensity was determined by flow cytometry (n = 3-4). (E) Cells were loaded with the Fluo-3AM, and calcium flux following SDF-1 stimulation (100 ng/mL) was monitored by flow cytometry (n = 4). (F,G) Adhesion assay: CFSE-labeled cells were pretreated with IgG or ALB6 for 4 hours and plated on (F) fibronectin-coated (n = 4) or (G) HUVEC-coated microwell plates (n = 4). Cells were either left unstimulated or stimulated with SDF-1 (100 ng/mL) for 45 minutes. Adherent cells from 5 random 20 × fields were counted under the Olympus IX71 fluorescence microscope. Data represent means ± SEM. *P ≤ .05; **P < .01.