Flow cytometric measurement of DCFH-oxidation of neutrophils in peripheral whole blood, isolated leukocytes and cerebrospinal fluid cells

Abstract

Using 2',7'-dichlorofluorescein diacetate (DCFH-DA) as an indicator for the intracellular formation of H2O2 and free radicals, we measured DCFH-oxidation (DCF fluorescence) of neutrophils by flow cytometry. The DCF fluorescence intensity of unstimulated neutrophils in whole blood was the same as the autofluorescence of neutrophils. However, that of unstimulated isolated neutrophils was higher than autofluorescence. The DCF fluorescence of isolated neutrophils was higher than that of neutrophils in whole blood with or without stimuli, and was decreased to the intensity of neutrophils in whole blood by the addition of plasma or erythrocytes. The DCF fluorescence of neutrophils in whole blood indicates the oxidative state of neutrophils in vivo. The determination of DCFH-oxidation of neutrophils in isolated leukocytes is also necessary to evaluate precisely the oxidative state of neutrophils. In cerebrospinal fluid (CSF) cells from patients with meningitis, the DCF fluorescence intensity of unstimulated neutrophils was higher than the autofluorescence of CSF neutrophils. The DCF fluorescence of CSF neutrophils stimulated with PMA showed increased intensity over that of unstimulated CSF neutrophils. The DCF fluorescence of CSF neutrophils, both unstimulated and stimulated with PMA, were decreased with the addition of plasma but not with the addition of CSF.