Effect of neutralizing anti-CXCR1 and anti-CXCR2 antibodies on CFU-MK growth and MK cell proliferation and differentiation in samples from healthy and MMM subjects. (A) CFU-MK colony formation from peripheral blood CD34⁺ cells of patients with MMM and healthy subjects. Purified CD34⁺ cells (5 × 103) were cultured in serum-free collagen medium with or without anti-CXCR1–neutralizing (black bar) or anti-CXCR2–neutralizing (gray bar) antibodies as described in “Materials and methods.” Colonies were scored at day 12 after staining with an anti-CD41 mAb–FITC and May-Grünwald-Giemsa. (B) (C) MK cell proliferation (panel B) and differentiation (panel C) from peripheral blood CD34⁺ cells of MMM and healthy subjects. Purified CD34⁺ cells were cultured for 12 days under megakaryocytic liquid culture conditions in the presence or absence of anti-CXCR1–neutralizing (black bar) or anti-CXCR2–neutralizing (gray bar) antibodies as described in “Materials and methods.” Cells were harvested after 14 days of culture, and cell numbers were determined by counting viable cells by trypan blue exclusion. After cells were labeled with monoclonal antibody against CD41, the percentage and MFI of CD41⁺ cells were analyzed with Cellquest software by means of a FASCcalibur flow cytometer. The expression level of CD41 and CD9 antigens on days 12 to 14 of culture compared with isotype control is expressed as MFI. In all experiments, results were expressed as the percentage above or below untreated control cells. Bar graphs display mean ± SEM from 3 independent experiments for normal blood cells. patients with MMM were individually analyzed. *P < .05. ND indicates not done.