Figure 1

PMA induced NET formation: A-M) Cells were treated for 4 h with PMA (A,B: control, C,D: 0.1 nM, E,F 1 nM, G,H 10 nM, I,J 100 nM, K,L 1 μM,) and microscopy performed at a density of 50,000 cells per well in a 96 well plate. Sytox Green cell impermeable dye was added at a concentration of 6 μM. Cells with a typical morphology of diffuse and spread NETs were noted [in this figure 50,000 cells were plated in 1 ml of media in plastic 24 well plates and stimulated as above with 100 nM PMA] (M). Scale bars = 10 microns. A greater abundance of NETs was seen with increases in PMA concentration but abundance was similar in the 10–1000 nM range. N) Fluorescence of extracellular DNA by cell impermeable Sytox Green was measured with an excitation of 485 nm and measured at 530 nm. Background fluorescence was subtracted and the abundance of NETS expressed as fluorescence in arbitrary units. The concentration-dependent response of NET formation plateaued after 10 nM PMA. Data show mean +/− SEM for n = 6 independent experiments.