Measurement of phagosomal pH of normal and CGD-Like human neutrophils by dual fluorescence flow cytometry

Background: Phagosomal pH is thought to play an important role in the antimicrobial activity of polymorphonuclear leukocytes (PMNs). In this study, we set up a method for a rapid and accurate measurement of phagosomal pH in PMNs with the use of Candida albicans doubly labeled with a pH-insensitive and a pH-sensitive probe and flow cytometry. Methods: Heat-killed, serum-opsonized C albicans were doubly labeled with fluorescein, a pH-sensitive probe, and rhodamine, a pH-insensitive probe, and incubated with human PMNs. Flow cytometric readings of PMN-associated Candida were then taken, and the intraphagosomal pH was calculated on the basis of the ratio of fluorescein: rhodamine fluorescence by using a calibration curve obtained after equilibration of phagosomal pH with different external pH values after addition of digitonin. Results: A rapid rise in phagosomal pH, which reached pH 7.8, was observed 2 min after initiation of phagocytosis and progressively declined to pH 6.9 after 15 min. Such a rise was not observed in PMNs with defective microbicidal activity (deficient in nicotinamide adenine dinucleotide phosphate oxidase), where phagosomal pH dropped to pH 6.6, 2 min after phagocytosis. The abnormal initial acidification in PMNs deficient in nicotinamide adenine dinucleotide phosphate oxidase was prevented by using lysosomotropic weak bases or the vacuolar-type H+ pump inhibitor concanamycin A. Conclusions: Phagosomal pH of PMNs can be easily and accurately measured by dual fluorescence flow cytometry. The method can be applied to assess phagosomal pH in PMNs with defective microbicidal activity and to monitor the outcome of pharmacologic interventions aimed at correcting its abnormalities. Cytometry 48:159-166, 2002. (C) 2002 Wiley-Liss, Inc.