Measurement of phagosomal pH of normal and CGD‐like human neutrophils by dual fluorescence flow cytometry
Open Access
- 27 June 2002
- Vol. 48 (3) , 159-166
- https://doi.org/10.1002/cyto.10123
Abstract
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.Keywords
Funding Information
- Italian Telethon Foundation (EC0533)
- IRCSS Burlo Garofalo (RC 31/98)
This publication has 25 references indexed in Scilit:
- Comparison of Three Common Amine Reactive Fluorescent Probes Used for Conjugation to Biomolecules by Capillary Zone ElectrophoresisBioconjugate Chemistry, 1995
- Folimycin (Concanamycin A), an Inhibitor of V-Type H+-ATPase, Blocks Cell-Surface Expression of Virus-Envelope GlycoproteinsBiochemical and Biophysical Research Communications, 1993
- Killing of pathogens associated with chronic granulomatous disease by the non-oxidative microbicidal mechanisms of human neutrophilsJournal of Medical Microbiology, 1991
- The inhibitory effects of some iodonium compounds on the superoxide generating system of neutrophils and their failure to inhibit diaphorase activityBiochemical Pharmacology, 1987
- The effect of the inhibitor diphenylene iodonium on the superoxide-generating system of neutrophils. Specific labelling of a component polypeptide of the oxidaseBiochemical Journal, 1986
- Endosome pH measured in single cells by dual fluorescence flow cytometry: rapid acidification of insulin to pH 6.The Journal of cell biology, 1984
- The measurement of superoxide anion production by granulocytes in whole blood. A clinical test for the evaluation of phagocyte function and serum opsonic capacityEuropean Journal of Clinical Investigation, 1983
- Binding reaction between the third human complement protein and small moleculesBiochemistry, 1981
- The respiratory burst of phagocytic cells is associated with a rise in vacuolar pHNature, 1981
- EndocytosisAnnual Review of Biochemistry, 1977