Journal of Leukocyte Biology 47:295-303 (1990) © 1990 Wiley-Liss, Inc. Oxidative Metabolism in the Alveolar Macrophage: Analysis by Flow Cytometry

We evaluated the reagents dichlorofluorescin (DCFH) and hydroethidine (HE) for use in flow cytometric analysis of the respiratory burst of alveolar macrophages and mono-cytes. DCFH and HE are non-fluorescent precursors which can be oxidized Intracellularly to the fluorescent compounds dichlorofluorescein and ethidium. Alveolar macrophages (AM5) loaded with either DCFH or HE were analyzed after phor-bol myristate acetate (PMA) stimulation. The results, expressed as fmol/cell oxidation product (DCF or ethidium) after fluorometric standardization of the flow cytometer, show that both DCFH (273 ± 48, mean Increase over control ± SE, fmol/cell, N = 9) and HE (416 ± 54, N = 11) detected the substantial respiratory burst of hamster AMs. Similar results were obtained with normal human AMs. By using multiparameter analyses, the oxidative response of AM5 ingesting opsonized fluorescent latex beads was measured in subpop-ulations ingesting Increasing numbers of particles. A graded increase In oxidation of both DCFH and HE was found in response to increasing phagocytosis. Ingestion of fluoresceinated staphylyococcal bacteria caused similar changes in HE-loaded AM5. In-hibition of respiration with antimycin showed that approximately 95 % of the increase