<i>In vitro</i> neutrophil phagocytosis, killing and NET formation.

<p>(A) A constant of killing was calculated using the method described by Hampton (MOI 1). The paired constants for each strain derived from 3 independent experiments are shown; differences between the two strains were significant (paired t-test, p = 0.01). (B) Fluorescent pneumococci (MOI 10) were cultured with mouse neutrophils and visualized by flow cytometry at the indicated times after bacterial addition. (C) as (B) but associated bacteria (closed bars, SRL1, open bars TIGR4) enumerated by fluorescence microscopy. Bars are means of at least 50 determinations; error bars are SEM. Differences between the strains are significant, p < 0.001, two-way ANOVA). (D-F) NET formation by neutrophils was assessed by fluorescence microscopy (MOI 10). (D) Representative confocal images of neutrophils forming NETs (blue: <i>S</i>. <i>pneumoniae</i>, green: anti-neutrophil elastase, red: sytox orange). (E) Percentage of neutrophils in NETs following incubation with indicated strains or PMA. Bars are means of at least 4 separate low power fields; error bars are SEM. Significant differences between the groups were determined by t tests with Tukey’s post hoc correction; ** p <0.01, *** p< 0.001. (F) mean NET size formed by TIGR4 and SRL1. Bars as in (E). Difference between the strains is significant, p < 0.05, t test. B-F representative of two independent experiments.