Figure 2
Mechanism of NET release to planktonic C. glabrata. (a) Production of ROS in response to C. glabrata (CG 006 at 3 × 106 cells/well) was measured by fluorescence after neutrophils were pre-stained with oxidative stress indicator CM-DCF and co-cultured with C. glabrata over 4 h. The mean and SEM of 4 experiments performed in triplicate on 4 occasions are shown. Data for each time point were analyzed by ANOVA (*P < 0.05), with pairwise comparisons using the Holm-Sidak method (**P < 0.05 for C. glabrata v. PMA). (b) Neutrophils were co-cultured with planktonic C. glabrata for various time points and NET production was estimated by Sytox Green. Results were normalized to the positive control, PMA, and data from 4 experiments performed in triplicate were combined, SEM shown. Data for each time point were analyzed by ANOVA (*P < 0.05 for pairwise comparison by Holm-Sidak). (c) The neutrophil response to planktonic C. glabrata was imaged with scanning electron microscopy at various time points over 4 h. Measurement bars represent 1 µm for 10,000x images. (d) Following co-culture with C. glabrata for 1 h or 4 h, samples were immunolabeled with an anti-citrullinated H4 antibody and a fluorescently labeled (DyLight) secondary antibody and examined with brightfield microscopy and fluorescent (565/620 nm) microscopy.
