CD44 is localized to the leading edge of polarized human HPCs migrating toward SDF-1. CB-derived CD34⁺ cells were plated on HA coverslips and allowed to adhere for 30 minutes. Cell movement was recorded as described in “Materials and methods.” The position of SDF-1 source is indicated by arrowheads. (A-C) Phase contrast microscopy of untreated cells (A), cells stimulated with polarized source of SDF-1 (B), and cells treated with anti-CD44 mAb F10-44-2 and stimulated with polarized source of SDF-1 (C). (D-F) Cells treated as above were fixed 5 and 30 minutes after exposure to polarized source of SDF-1 and indirectly immunolabeled with antihuman CD44 mAb (red) and anti-CXCR4 mAb (green). Projected images of consecutive optical sections through the cell volume are shown. An arrow is pointing to the fine CD44-positive protrusions at the direction of SDF-1. Panels Eii and Fii are images obtained from a single optical plane close to the HA-coated surface of cells depicted in panels Ei and Fi, respectively, showing preferential CD44 (red) localization to the leading edge (original magnification, × 160 for panels Eii and Fii). In panel Fii, only part of the membrane located at the cell front is labeled by anti-CD44. Bars = 5 μm.