Mobilized CD34⁺ PBPCs express a CXCR4 molecule truncated in the first extracellular domain. (a) Nalm-6 cells were incubated for 2 hours at 37°C in the presence of indicated concentrations of purified NE (circles) or CG (squares) and were stained with the mAbs 6H8 or 12G5. After analysis by flow cytometry, results are expressed as a percentage of 6H8/12G5 binding of nontreated cells. Data represent means ± SD of three independent experiments. (b) Binding of 6H8 and 12G5 mAbs to steady-state CD34⁺ BM cells, GCSF–mobilized CD34⁺ PBPCs, and GCSF–mobilized CD34⁺ PBPCs after overnight culture. The flow cytometry analysis was gated on CD34⁺ cells. Representative data from two experiments are shown. (c) CD34⁺ cells isolated from normal steady-state BM were treated with 100 μg/ml of NE or CG. Control cells were pretreated in an identical manner with PBS in the absence of protease. The chemotactic response of cells was assessed in the presence (black bars) or absence (white bars) of 200 ng/ml of CXCL12 in the lower chamber. Representative data from two experiments in triplicate are shown. P, PBS. (d) Freshly isolated GCSF–mobilized CD34⁺ PBPCs or CD34⁺ cells derived from steady-state BM were compared for their chemotactic response in the absence (white bars) and presence (black bars) of 200 ng/ml CXCL12. Representative data from two experiments are shown.