Neutrophil Extracellular Trap Killing Assay of Candida albicans.

Fungal pathogen Candida albicans is one of the top leading causes of overall healthcare-associated bloodstream infections worldwide. Neutrophil is the major effector cell to clear C. albicans infection. Our study showed that mouse neutrophils utilize two independent mechanisms to kill C. albicans: one is CR3 downstream NADPH oxidase-dependent mechanism that kills opsonized C. albicans; the other one is dectin-2-mediated NADPH oxidase-independent neutrophil extracellular trap (NET) that kills unopsonized C. albicans. Neutrophil killing of opsonized C. albicans requires phagocytosing the organism and production of reactive oxygen species production (ROS). Most existing protocols that assay for neutrophil killing of C. albicans requires a washing step after allowing neutrophils to phagocytose the organism. By definition, NET kills organisms extracellularly. Therefore, it is important to skip the washing step and add an optimal ratio of neutrophils and C. albicans to the wells. To demonstrate the effect of NET, it is necessary to compare killing ability of neutrophils treated with micrococcal nuclease (MNase), an enzyme that digests NET, to that treated with heat-inactivated MNase. MNase is also applied to release NET-bound fungal elements for counting. This protocol can be applied to assay NET killing of other biofilm-forming organisms.