PDK2/4^–/– neutrophils exhibit reduced release of NETs. NETs assay was performed by stimulating bone marrow–derived WT and PDK2/4^–/– neutrophils with PMA (10 nM) for 4 hours. Representative microphotographs of NETs stained with Sytox green (stains extracellular DNA, green) and counterstained with Hoechst (stains nuclei, blue) are shown. The bar graph shows the quantification of the percentage of cells releasing NETs. Values are mean ± SEM, n = 5 mice per group. Two different fields at 20× magnification were used to estimate the number of neutrophils releasing NETs, which was expressed as a percentage of total neutrophils (coverslip edges were avoided). Statistical analysis was performed using 2-way analysis of variance (ANOVA) followed by Tukey multiple comparisons test.