Chemotaxis of cells to an SDF-1 gradient is lipid raft dependent. (A) Chemotaxis of CD34⁺ cells to medium alone (control), SDF-1 (300 ng/mL), and SDF-1 (300 ng/mL) after preincubation for 1 hour with amphotericin (10 μg/mL) or nystatin (50 μg/mL). Cells were collected after chemotaxis from the lower chambers and were plated in clonogenic assay. The number of CFU-GM and BFU-E colonies formed by harvested cells is shown as a percentage of control values. Data are pooled from quadruplicate samples from 3 independent experiments. *P < .0001. (B) Chemotaxis of CD34⁺ cells toward medium alone (control), SDF-1 low (10 ng/mL) alone, or SDF-1 low with cells exposed to fibronectin (2 μg/mL) or fibrinogen (4 μg/mL). Cells pretreated before chemotaxis for 1 hour with MβCD (2.5 mM) are shown as red bars. Data are pooled from quadruplicate samples from 3 independent experiments. *P < .00001. (C) Lipid raft formation on CD34⁺ cells. mPB CD34⁺ cells were kept in SLPs (lower panel) or were washed out of SLPs (upper panel) and resuspended in control medium. Primary antibodies used for raft analysis were cholera toxin β-subunit conjugated with FITC and mouse monoclonal anti-hCXCR4 IgG. Stained cells were examined using a BX51 fluorescence microscope (Olympus America, Melville, NY) equipped with a charge-coupled device camera (Olympus America). Separate pictures are merged using Image-Pro Plus software (Media Cybernetics Inc., Silver Spring, MD). Lipid raft formation was analyzed on samples from 3 patients who underwent mobilization with G-CSF. Results from a representative study are shown. Colocalization of GM1 and CXCR4 is shown as yellow patchy staining. Magnification × 60.