Fig 1: S100A8/A9 is not released during exocytosis. (A) Neutrophils (5 × 105) from healthy controls were left unstimulated or incubated with PAF (1 µM, 5 min) or cytochalasin B (5 µg/mL, 5 min) and subsequently stimulated with fMLF (1 µM) for 10 min at 37 °C. To obtain the cytoplasmic fraction of neutrophils, digitonin (150 µM, 15 min) was used to permeabilize the plasma membrane of neutrophils. As a positive control, neutrophils were permeabilized with Triton X-100 (1%, 10 min). S100A8/A9 concentration was determined by ELISA (n = 4–6, mean + SEM). (B) Experiment to determine the optimal concentration of digitonin to isolate the cytoplasmic fraction. Lactate dehydrogenase (LDH) levels and protease levels were measured and are presented as percentage of their total content (n = 1). (C) Panel I: Lactoferrin concentration in supernatant of unstimulated, PAF/fMLF-stimulated neutrophils, or the cytoplasmic fraction of neutrophils was determined by ELISA (n = 2, mean + SEM). Panel II: Myeloperoxidase concentration in supernatant of unstimulated or CytoB/fMLF-stimulated neutrophils was determined by ELISA (n = 2, mean + SEM). (D) Presence of DNA was measured with Sytox Green Nucleic Acid Stain (n = 3, mean + SEM). (E) Representative image of digitonin-treated neutrophils. DNA was visualized with Hoechst (blue, 405). Images were acquired using a Leica SP8 confocal microscope, magnification 630x, scale bar = 10 µm. (F) Coomassie Brilliant Blue staining and Western Blot for S100A8 (red) and S100A9 (green) of the cytoplasmic fraction of neutrophils. (G) Representative immuno-electron microscopy image of a neutrophil (right) and lymphocyte (left) stained for anti-S100A8 (black dots). Scale bar = 1 µm. “A” indicates azurophilic granules and “S” indicates specific granules. S100A8 is not present in neutrophil granules. Statistics were performed by mixed effects’ model (A) or one-way ANOVA (D) with Dunnett post hoc test; ** p < 0.01, *** p < 0.001, ns = not significant; n.d. = not detectable.