SDF-1–induced actin polymerization and calcium (Ca²⁺) flux in CD34⁺ cells. (A) Ex vivo–expanded CD34⁺ cells and (B) nonexpanded CD34⁺ cells were pretreated with TGF-β1 (0.5 ng/mL), SDF-1 (200 ng/mL), SDF-1 (200 ng/mL) + TGF-β1 (0.5 ng/mL), or cytokine cocktail alone (medium) for 20 to 24 hours. Cells were then washed and stimulated with 200 ng/mL SDF-1 for the indicated times to induce actin polymerization. Cells were stained with FITC-phalloidin and then subjected to flow cytometry. Data represent the mean ± SD of 3 independent experiments. *P < .05 compared with CD34⁺ cells pretreated with medium, TGF-1, or SDF-1 + TGF-β1. SDF-1–mediated calcium flux in (C) ex vivo–expanded CD34⁺ cells and (D) nonexpanded CD34⁺ cells pretreated with medium alone, TGF-β1 (0.5 ng/mL), SDF-1 (200 ng/mL), or TGF-β1 (0.5 ng/mL) along with SDF-1 (200 ng/mL). Cells were loaded with Fluo-3AM. Baseline fluorescence was first recorded and then calcium flux following SDF-1 (200 ng/mL) stimulation (indicated by ↑) was analyzed by FACScan. Increase in fluorescence intensity following SDF-1 addition (compared with baseline) represented the calcium flux in the cells and is shown on the y-axis of the plot. Data represent the mean ± SEM of 3 independent experiments. *P < .05 compared with cells pretreated in cytokine cocktail alone or along with TGF-β1.