Fig. 3.

LL-37 rapidly converts neutrophils from AV⁺PI⁻ to AV⁺PI⁺ with the release of cellular contents. Fresh neutrophils were aged in culture for 18 h (60% AV⁺PI⁻) and treated with LL-37 (0–5 μM), and the corrected number (see Fig. 1 for details of the calculation) of AV⁻PI⁻ and AV⁺PI⁻ cells was measured over time after treatment (a and b). Eighteen-hour-aged neutrophils were mixed with FITC-AV and PI, and time-lapse photography was performed on an inverted fluorescent microscope equipped with a 37°C incubator stage. Sequential images of phase contrasts and red and green channels were captured at a rate of three frames per minute over a period of 10 min after the addition of LL-37 (5 μM). The combined phase and green or red channels are shown for the untreated (c, upper) and treated cells (c, lower) after 10 min. Eighteen-hour-aged neutrophils were also labeled with CFSE, treated with LL-37 for 5 min, and stained with APC-labeled AV, and the corrected number of AV⁻CFSE⁺ (living; d) and AV⁺CFSE⁺ (apoptotic) cells (e) was measured. (a–e) n = 4; *, P < 0.05; **, P < 0.01; ***, P < 0.001, versus untreated controls. Fresh (open bars) or 18-h-aged AV⁺ (solid bars) neutrophils were incubated with LL-37 (0–5 μM) for 5 min, and the levels of LDH released into the medium were measured (f). Lys, Cell lysate; n = 4, *, P < 0.05; **, P < 0.01; ***, P < 0.001, versus untreated control of fresh neutrophils; ^##, P < 0.01, versus untreated control of AV⁺ cell. RFU, Relative fluorescence units.