Abstract
Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.
Highlights
The neutrophil is the most abundant innate immune cell in the body
Its main function consists of patrolling the bloodstream and engulfing and digesting the foreign particles that it may encounter in a process known as phagocytosis[1,2]
The activation of neutrophil NADPH oxidase, the isoform NOX2, initiates a cascade of biochemical reactions that culminates in the death of the pathogen
Summary
The neutrophil is the most abundant innate immune cell in the body. Its main function consists of patrolling the bloodstream and engulfing and digesting the foreign particles that it may encounter in a process known as phagocytosis[1,2]. It transports electrons from NADPH across the membrane to molecular oxygen inside the phagosome, producing superoxide anions and further reactive oxygen species This is known as the respiratory burst, and it is thought to be essential for efficient microbial killing and digestion[2]. Proton concentration is reflected by pH, so for a given level of oxidase activity, measuring the pH in the phagosome can provide information on the relative participation of protonic and non-protonic pathways in charge compensation. The human neutrophil phagosome has an alkaline pH of approximately 8.5 for 20-30 min after phagocytosis[5] This implies the existence of additional non-proton ion channels in NOX2-induced charge compensation, as the fusion and release of the contents of the acidic granules and sole compensation by HVCN1 would maintain an acidic environment, in contrast to that observed. NOTE: Use tubes with low surface binding material, if possible, as HKC-S-1 particles can stick to the wall of normal centrifuge tubes
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