Figure 5

Mechanism of NET induction by planktonic and biofilm C. glabrata. (a) Production of ROS in response to C. glabrata was measured by fluorescence after neutrophils were pre-stained with oxidative stress indicator CM-H2DCFDA and co-cultured with C. glabrata over 4 h. The mean and SEM of 4 experiments performed in triplicate is shown. The statistical significance neutrophil production of ROS in response to biofilm and planktonic C. glabrata were calculated for each time point using a two-tailed Student’s t-test assuming unequal variances, *P < 0.05, SEM shown. (b) Neutrophils were treated with DPI to inhibit NADPH-oxidase and the release of NETs in response to C. glabrata was measured by Sytox Green. The percent of the total free DNA (untreated) reduced by DPI-treatment for each condition is shown. Data represent 5 experiments performed in triplicate. Statistical significance was determined using a Student’s t-test compared to no inhibition, *P < 0.05, SEM shown. (c) Neutrophil-C. glabrata interactions after 4 h were imaged with scanning electron microscopy. (d) Calcein AM-labeled neutrophils (green) were treated with cytochalasin D to inhibit phagocytosis and added to planktonic C. glabrata. Neutrophil interactions were imaged at 1 h (40x). (e) Neutrophils were treated with cytochalasin D to inhibit phagocytosis and the release of NETs in response to C. glabrata was measured by Sytox Green. The percent of the total free DNA (untreated) reduced by DPI treatment for each condition is shown. Data represent 5 experiments performed in triplicate. Statistical significance was determined using a Student’s t-test compared to no inhibition, *P < 0.05, SEM shown.