Effects of LL-37 on neutrophil apoptosis and caspase 3 activity. Neutrophils (10⁶ cells/mL) were incubated for 18 h at 37°C in RPMI1640-10% FBS in the absence (Control) or presence of LL-37 ( μg/mL) or LPS (10 ng/mL). Neutrophils were also incubated for 18 h at 4°C in the absence of LL-37 or LPS (Resting). After incubation, neutrophils were cytocentrifuged and stained with May-Grünwald-Giemsa, and a minimum of 300 neutrophils/slide was examined by light microscopy on duplicate cytospins. Apoptotic neutrophils were identified based on the morphological changes (such as chromatin condensation, rounded nuclear profiles, cell shrinking, membrane blebbing, and cytoplasmic vacuolization), quantitated and expressed as a percentage of apoptotic cells (a). Alternatively, caspase 3 activity was assayed by incubating neutrophil lysates with acetyl-Asp-Glu-Val-Asp-p-nitroanilide substrate in the absence or presence of acetyl-Asp-Glu-Val-Asp-al, a specific caspase 3 inhibitor at 37°C for 2 h. Caspase 3 activity is expressed as nmol of p-nitroanilide liberated/10⁶ cells/h (b). Data are the mean ± SD of 4 to 18 separate experiments. Values are compared between the incubation at 37°C in the absence (Control) and presence of LL-37 or LPS. All the data in this paper are analyzed for significant difference by a one-way analysis of variance (ANOVA) with multiple comparison test (Prism 4, GraphPad Software, San Diego, CA, USA). *; ***.