When neutrophils engulf bacteria they enclose them in small vacuoles (phagosomes) into which superoxide is released by activated NADPH oxidase (NOX2) on the internalized neutrophil membrane. The directional nature of NOX2 activity creates a charge imbalance that must be counteracted to prevent depolarization of the membrane and the shutdown of activity (Winterbourn CC et al. 2016). Also, protons are produced in the cytosol and consumed in the external compartment (for example, the phagosome) through the dismutation of superoxide. Both situations are largely overcome by a balancing flow of protons transported by voltage-gated proton channels, primarily VSOP/HV1, which are activated in parallel with the oxidase (Demaurex N & El Chemaly A 2010; El Chemaly A et al. 2010; Petheo GL et al. 2010; Kovacs I et al. 2014; Henderson LM et al. 1987, 1988). The pH of the phagosome is regulated by these activities. In contrast to the phagosomes of macrophages, in which pH drops following particle ingestion, neutrophil phagosomes remain alkaline during the period that the oxidase is active. Until recently, their pH has been accepted to lie between 7.5 and 8. However, in a 2015 study using a probe that is more sensitive at higher pH, an average pH closer to 9 was measured in individual phagosomes (Levine AP et al. 2015).

The superoxide dismutates to hydrogen peroxide, which is used by myeloperoxidase (MPO) to generate other oxidants, including the highly microbicidal species such as hypochlorous acid (Winterbourn CC et al. 2013, 2016).