Figure 1. Extracellular chloride (Cl⁻) contributes to bacterial killing by human neutrophils.

(A) Human peripheral neutrophils were incubated in minimal media with or without Cl⁻ for 3 h (2×10⁶ cells/well). Cells were then assayed for viability using calcein (green) which identifies live cells, and ethidium homodimer (red) which binds DNA but cannot cross intact cell membranes, thus staining dead cells. Neutrophils incubated for 3 h in minimal media with and without Cl⁻ are equally viable. (n = 3 wells per condition) (B) Human peripheral neutrophils were incubated in minimal media with or without Cl⁻. Bacteria were incubated in same media, with and without neutrophils (MOI  = 0.1). Bacterial killing by neutrophils was reduced in the absence of extracellular Cl⁻. Each condition performed in triplicate. (C) Oxidative burst was assayed using intracellular DCF dye that fluoresces when oxidized. The production of oxidants was slower in the absence of Cl⁻, but similar peak production of oxidants was achieved by 100 min in the presence or absence of Cl⁻. Each condition was done in triplicate in each experiment (D) Oxidant production in activated neutrophils occurs in a series of reactions. Enzymes known to be important in these reactions, such as NADPH oxidase and MPO are also essential for NETosis.