SDF-1 induces adhesion of CD34⁺ cells to fibronectin. (A) Ex vivo–expanded CD34⁺ cells were pretreated with cytokine cocktail alone (medium) or containing TGF-β1 (0.5 ng/mL), SDF-1 (200 ng/mL), or SDF-1 (200 ng/mL) + TGF-β1 (0.5 ng/mL) for 24 hours. The cells were washed and then plated on fibronectin (10 μg/mL) coated wells and incubated for 30 minutes. Cells were either left unstimulated (□) or were stimulated with SDF-1 (200 ng/mL) for the full 30 minutes (▪) or only for the last 2 minutes (▩) of the adhesion assay. Data represent the mean ± SD of 3 independent experiments. *P < .05 compared with adhesion response in absence of SDF-1 stimulation (basal adhesion) for respective pretreatment and **P < .05 compared with cells pretreated with SDF-1 alone. (B) CD34⁺ cells pretreated under various conditions were preincubated with anti–VLA-4 and anti–VLA-5 antibodies and assayed in adhesion assay. The cells were plated in fibronectin-coated plates and stimulated with SDF-1 for 30 minutes. Data represent the mean ± SD of 3 independent experiments. *P < .05 compared with adhesion response in the absence of antibodies. (C) Nonexpanded CD34⁺-enriched cord blood cells were cultured in medium (containing 20% FCS) alone or along with TGF-β1 (0.5 ng/mL), SDF-1 (200 ng/mL), or SDF-1 (200 ng/mL) and TGF-β1 (0.5 ng/mL) for 24 hours. These cells were then assayed for their ability to bind to fibronectin in the absence or presence of 200 ng/mL SDF-1 for 2 minutes (transient response) and 30 minutes (sustained response). Nonadherent cells were removed, and adherent cells were quantified as described in “Materials and methods.” Data represent the mean ± SEM of 3 independent experiments. *P < .05 compared with adhesion response in absence of SDF-1 stimulation (basal adhesion) for respective pretreatment. (D) Fresh CD34⁺ cells pretreated under various conditions for 24 hours were preincubated with anti–VLA-4 antibody and assayed in adhesion assay as described in “Materials and methods.” Data represent mean ± SEM of 1 experiment. *P < .05 compared with adhesion response in absence of antibodies.