Effects of G-CSF on CXCR4 expression in human bone marrow myeloid cells. (A-B) Unfractionated bone marrow cell populations were incubated (1 × 10⁶/mL) at 37°C for 18 hours in medium alone or with G-CSF (1 and 100 ng/mL). Cells were double stained for CD33 (PE-labeled) and CXCR4 (APC-labeled 12G5 antibody) or with PE- and APC-labeled control antibodies (isotype). (A) Surface CXCR4 expression in human bone marrow CD33⁺ myeloid cells cultured for 18 hours in medium alone (none) or with G-CSF (1 and 100 ng/mL). (B) Surface CXCR4 expression in human bone marrow CD33^– cell populations cultured for 18 hours in medium alone (none) or with G-CSF (1 and 100 ng/mL). (C) Expression of surface CXCR4 in human CD33⁺ bone marrow cells as a function of culture time (0-18 hours) in medium alone or with G-CSF (100 ng/mL). The results are expressed as mean fluorescence intensity (MFI) of CXCR4 detected on CD33⁺ cells (mean ± SEM of 3 experiments). (D) Time-dependent reduction of surface CXCR4 in CD33⁺ cells induced by G-CSF (100 ng/mL). Unfractionated bone marrow cell populations were incubated at 37°C for 18 hours with or without G-CSF (100 ng/mL). The results are expressed as the mean (± SEM of 3 experiments) percent reduction of surface CXCR4 induced by G-CSF compared with medium alone. *P < .05 medium versus G-CSF. (E) CXCR4 detected in cell lysates of purified bone marrow CD33⁺ cells (> 85% purity) immediately after separation (0h), and after 1.5-hour and 18-hour incubation in medium only (M) or with G-CSF (G, 100 ng/mL). Bottom panel reflects the results upon membrane reprobing with anti–beta actin antibodies.