Figure 4.

NET release by neutrophils exposed to C. albicans clinical strains. (a) Neutrophils were co-cultured with planktonic cells or biofilms for 4 h, and NET release was measured with a Sytox Green fluorescence assay. Neutrophils alone (-) served as a negative control, and neutrophils stimulated with PMA were included as a positive control and for normalization. The release of NETs upon encounter with biofilm and planktonic organisms for each strain was compared using a Student’s t-test, * p < 0.05, n = 5, SEM shown; (b) In order to visualize free DNA following neutrophil co-culture with planktonic C. albicans, neutrophils were pre-stained with Calcein AM (green) and co-incubated with planktonic cells (SC5314) for 4 h. After staining with propidium iodide (red), images were taken at 20×, and digitally enlarged 2× for comparison; (c) To assess the degree of histone citrullination, SC5314 planktonic co-cultures were stained with anti-H4 citrulline 3 antibody (red). Images were obtained at 40×.