Figure 6

Flow cytometry of cultured vascular smooth muscle cells. Dot-plots (left) of FITC-annexin V (x-axis)/propidium iodide (y-axis) fluorescence and corresponding histograms for FITC-annexin V fluorescence (right) of vascular smooth muscle cells incubated in the absence (A, B, control) or presence of LL-37 (30 μg/ml) for 10 min (C, D), 30 min (E, F), 60 min (G, H) or 180 min (I, J). The diagrams on the left are divided into four areas and the percentages of the total number of stained cells within each area are given. Viable control cells are found in the lower left part if the diagrams and non-viable cells, heavily stained with FITC-annexin V and propidium iodide, are found in the upper right part of the diagrams. Apoptotic cells, stained with FITC-annexin V but not propidium iodide, are found in the lower right part of the diagrams. Incubation with LL-37 resulted, within 10 min, in a moderate increase in propidium iodide staining (C) of the viable cells followed by a gradually developing increase in FITC annexin V staining from 5.1 % at 10 minutes to 28 % at 180 minutes indicating the development of apoptosis (E-J). The fraction of non-viable cells increased from 17 to 54 % during the whole time course studied.