Evaluation of human monocyte oxidative metabolism utilizing a flow cytometric assay

Assays routinely employed to evaluate the human monocyte respiratory burst activation may have been limited to measuring responses of bulk cell preparations. We demonstrate that individual monocyte responses can be easily assessed by using 2',5' dichlorofluorescin diacetate (DCFH-DA) and flow cytometry. Adherence purified monocytes were incubated with DCFH-DA, washed, and stimulated with either phorbol myristate acetate (PMA) or heat-aggregated IgG (HAIgG). Log green fluorescence signals were measured by using a flow cytometer equipped with a 5-W argon laser set at an excitation wavelength of 488 nm. Optimal conditions for stimulation included exposure to 5 μM concentrations of DCFH-DA for 15 min, followed by a 60-min incubation with either PMA or HAIgG. Dichlorofluorescin (DCFH) oxidation by monocytes increased in a graded fashion as a function of stimulus concentration. Monocytes responded as a uniform population in response to increasing doses of PMA and HAIgG. This oxidative response was also monitored in mixed populations of mononuclear leukocytes, with monocytes identified on the basis of light scatter properties and surface antigen staining with anti-CD14. More than 90% of cells demonstrating increases in log green fluorescence signals following activation were CD14 positive. Measurement of DCFH oxidation by monocytes is reflective of the capacity to undergo a respiratory burst response, in that monocytes obtained from patients with chronic granulomatous disease were only minimally reactive. This assay, representing a rapid means of assessing monocyte respiratory burst activation by single cell analysis, is suitable for use in both clinical and research settings.