Cross-linking of Siglec-9 using different techniques excludes the possibility of nonspecific death effects. (A) F(ab')₂ fragments of the anti-Siglec-9 mAb and intact mAb revealed same results. Cross-linking by a plate-bound primary anti-Siglec-9 mAb also resulted in neutrophil death. GM-CSF was under these 3 different conditions always an efficient death sensitizer. F(ab) fragments of the anti-Siglec-9 mAb had no cytotoxic effect. Moreover, preincubation of F(ab) fragments of the anti-Siglec-9 mAb prevented neutrophil death induced by the intact mAb both in the presence and absence of GM-CSF. Results of 20-hour cultures are shown (n = 4). ^***P < .001. Data in panel A are expressed as means ± SEM. (B,C) Anti-Siglec-9 mAb stimulation in the absence of GM-CSF-induced PS redistribution (9-hour cultures) and DNA fragmentation (13-hour cultures) indicative for the induction of neutrophil apoptosis. In presence of GM-CSF, Siglec-9 ligation resulted in even more DNA fragmentation, but an abnormal subpopulation of neutrophils with low annexin-binding capacity was observed. These panels show one representative experiment out of 5. In panel B, quantitative analysis is shown in percent at the top right corner of each dot plot. In panel C, the relative number of apoptotic cells is indicated by the bracket in each subpanel.