Secretion of SDF-1 by cultured CD34⁺ cells. (A) Determination of SDF-1 Ag in CD34⁺ cell supernatant by ELISA. CD34⁺ cells (2 × 10⁵/mL) were cultured for 5 days in serum-free medium in the presence of cytokines. Cell supernatants were obtained from: different donors (A through G) and sources (UCB, BM, MPB) of CD34⁺ cells; sorted MPB CD34⁺/CD38⁺ cells; a human polyclonal T-cell line (PBL); the mouse stromal cell line MS-5. Supernatants were assayed for the presence of SDF-1 by an Ag capture ELISA, as indicated in Materials and Methods. (B) Migration of CD34⁺ cells in response to serum-free supernatant from UCB CD34⁺ cells cultured in the presence of IL-3, SCF, and IL-6 for 4 days and 7 days, respectively. Chemotaxis to serum-free medium containing cytokines (assay medium) and to low doses of SDF-1 (10 ng/mL and 30 ng/mL) are shown as controls. (C) Inhibition of chemotaxis induced by supernatant from CD34⁺ cells. Chemotaxis of CD34⁺ cells in response to 5 days supernatant from UCB CD34⁺ cells in the presence of an anti-CXCR4 MoAb or of a control MoAb.