Figure 1.

Effect of Cl-amidine on NET induction in vitro. Human peripheral blood neutrophils were seeded in wells of 4-well chamber slides (1 × 10⁶/ml). After incubation for 30 min at 37°C, the cells were exposed to 0 or 20 nM PMA with or without 20 μM PTU. Fifteen minutes prior to PMA/PTU administration, 200 μM Cl-amidine was added alternately into the wells. After 2 h of incubation at 37°C, the medium containing the reagents was removed, and the remaining cells were washed with PBS followed by fixation with 4% paraformaldehyde for 15 min. Thereafter, immunofluorescent staining for citrullinated histone 3 was carried out followed by mounting with the DAPI-containing solution. Representative photos (A). Red, citrullinated histone 3; Blue: DNA (original magnification: ×400). Quantification of NET formation (B). NET formation was quantified by counting the citrullinated histone 3-positive cells per × 100 power field of view. Data from five random fields of view (×100) were subjected to quantitative analysis. **p < 0.01 in Mann–Whitney U-test.