FIG. 7.

Polymyxin B-treated LOS induces NET formation to lower levels than those induced by untreated LOS. Neutrophils (10⁶/well) were seeded into wells of a chamber glass coverslip and allowed to adhere. Wells remained untreated or were treated with either 0.032 ng/μl NTHI LOS, 0.032 ng/μl NTHI LOS pretreated with 50 μg/ml polymyxin B for 20 min, or an equivalent amount of polymyxin B alone (∼0.53 μg/ml). After 5 h of incubation, samples were processed for immunofluorescence as described in the text and viewed using immunofluorescence confocal microscopy. At least 65 randomly selected fields of view from four wells per treatment condition and staining condition were observed to quantify the percentage of viewing fields positive for NET formation, based on colocalization of DNA with either histone (left) or neutrophil elastase (right). White bars, untreated LOS; light gray bars, polymyxin B-treated LOS; dark gray bars, polymyxin B alone. Data were obtained from three separate experiments, and statistical significance was determined by a nonparametric t test. *, P < 0.05.