Assessment of neutrophil apoptosis by the morphological changes and FITC-annexinV/propidium iodide-staining. Neutrophils (10⁶ cells/mL) were incubated for 18 h at 37°C in RPMIFBS in the absence ((b) and (e)) or presence of LL-37 (1 μg/mL; (c) and (f)). Neutrophils were also incubated for 18 h at 4°C in the absence of LL-37 ((a) and (d)). After incubation, neutrophils were stained with May-Grünwald-Giemsa and FITC-annexinV/propidium iodide [23–25], and neutrophil apoptosis was assessed by the morphological changes (a–c) and flow cytometry (d–f), respectively. Apoptotic neutrophils exhibit characteristic features of chromatin condensation, formation of rounded nuclear profiles, cell shrinking, and presence of cytoplasmic vacuolization (as indicated by arrowheads), and were defined as annexin V-positive but propidium iodide-negative cells. Representative data are shown.