Rapamycin inhibits NET formation by human PMNs. (A) NET formation was assessed by live cell imaging with confocal microscopy (magnification, ×20). Extracellular and intracellular DNA were detected with a combination of cell permeable and cell impermeable DNA dyes (grayscale). Yellow arrows highlight areas of NET formation. Human PMNs were stimulated with LPS (100 ng/mL) for 1 hour with or without pretreatment with rapamycin (200nM) or FK-506 (200nM) as a control for rapamycin. These images are representative of visual fields selected from randomly captured fields from assays performed with PMNs from 5 different adult donors. (B) We quantified extracellular histone H3 content in DNase-treated supernatants by Western blotting as a surrogate for NET formation by human PMNs. Human PMNs were stimulated as above. The image is representative of assays performed with human PMNs isolated from 4 different healthy adult donors with densitometry analysis (n = 4) indicated in the graph below. Columns represent mean ± SEM supernatant histone H3 fluorescent intensity. The Kruskal-Wallis equality-of-populations rank test with 2-sample Wilcoxon rank sum posthoc testing was used. *Statistical significance with P < .05 in comparison with both control and LPS/rapamycin samples.