Highly purified preparations of human neutrophils produce the soluble GM-CSF receptor.

110 mL of supernatant conditioned overnight by freshly isolated human neutrophils (1 × 10⁷ neutrophils/mL) were applied to a GM-CSF ligand affinity column and eluted fractions were pooled, dialyzed against distilled H₂O lyophilized, and resuspended in 500 μL PBS. Panel A: 25 μL of the pooled sample was used in¹²⁵I-GM-CSF solution phase binding assays in the absence or presence of a large excess of unlabeled GM-CSF. Specifically bound radioactivity (spec bound, cpm) was calculated by subtracting the precipitated radioactivity in the absence of unlabeled GM-CSF from that in the presence of unlabeled GM-CSF. Bars represent the mean and SEM of duplicate experiments. Panel B: 30 μL of the pooled sample was subjected to 10.5% SDS PAGE under reducing conditions and transferred to PVDF membrane. Immunoblotting was performed with anti-GMRα antibody 8G6. Positive control (+ve) was ligand affinity purified recombinant solGMRα. Negative control (-ve) was sham ligand affinity column eluate.