sP-selectin must dimerize or oligomerize to induce leukocyte signaling in vitro. (A-B) Static adhesion of mouse leukocytes to immobilized ICAM-1 or fibrinogen, in the presence of the indicated concentration of sP-selectin or sP-selectin-Fc with or without blocking anti-P-selectin mAb. (C) Fluorescence micrographs of purified mouse neutrophils incubated with PMA (positive control, top row); with buffer (Untreated), sP-selectin, sP-selectin-Fc, or control IgG (Control-Fc) (middle 2 rows); or with sP-selectin or sP-selectin-Fc oligomerized with mAb HPC4 in the presence or absence of neutralizing anti-P-selectin mAb (bottom 2 rows). After incubation, the cells were fixed and stained with anticitrullinated histone IgG or with Sytox Green to label DNA. The insets in the top row are higher magnifications. The red arrow marks a neutrophil without staining for citrullinated histones and with staining for DNA only in the nucleus. The white arrow marks a neutrophil stained for citrullinated histones and DNA only in the nucleus. The yellow arrowhead marks a neutrophil with extracellular staining for both citrullinated histones and DNA, indicating NET release. Original magnification ×20; inset magnification 2.7-fold. Scale bar, 50 μm. (D-E) Quantification of cells stained for citrullinated histones or forming NETs (stained for both citrullinated histones and extracellular DNA). The data in panels A, B, D, and E represent the mean ± SEM from 5 experiments. *P < .05, **P < .01, ***P < .001.