Simultaneous measurement of phagocytosis and phagosomal pH by flow cytometry: Role of polymorphonuclear neutrophilic leukocyte granules in phagosome acidification

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Abstract
Human polymorphonuclear neutrophilic leukocytes (PMNLs) phagocytosed fluorescein‐isothiocyanate (FITC)‐labelled Staphy‐lococcus aureus. Free bacteria, phagocytes, and nonphagocytes were discriminated and quantified by flow cytometry (FCM). The relative fluorescence of phagocyte‐associated and free bacteria (Nf:N) was calculated by dividing the mean phagocyte fluorescence by that of the free bacteria and the number of phagocytosed bacteria. Bactericidal capacity and chemiluminescence were measured by standard methods. The red‐to‐green fluorescence ratio of acridine orange‐stained PMNLs (R/G) was measured by FCM. Degradation of bacteria was monitored by the reduction in FITC and ethidiumbromide fluorescence of bacteria liberated from the phagocytes. Bacterial FITC fluorscence was pH dependent. Nf:N was 0.5 to 0.7. Using a standard curve for the interrelationship between bacterial fluorescence and pH, phagosomal pH was 5.0–5.5 Phagocytes, kept at 4°C for 24 h had Nf:N ∼ 1, did not degrade bacteria, but killed them and emitted chemiluminescence. NH4Cl increased phagocyte fluorescence by 27% and decreased R/G by 50%. Cyanide and azide did not affect Nf:N. Nf:N of phagocytes from a patient with chronic granulomatous disease was 32% below, and R/G was 32% higher than the controls. Acidification of the phagosomes seems to be related to discharge of PMNL granule contents and independent of the respiratory burst.