Abstract
H2O2 production by neutrophils was measured by single cell analysis using flow cytometry (Epics-C) . This method is very useful for clinical evaluation of patients especially in pediatrics, because it requires only a little volume of blood (about 0.1 ml) and time less than an hour.
Cells were preincubated for 15 min with 5μM 2', 7'-dichlorofluorescin diacetate, which diffused into cells and were trapped within the cells. The cells, added stimulant such as phorbol myristate acetate, were incubated another 30 min. After lysing red blood cells by hypotonic shock, cells were washed and resuspended into PBS, and introduced to flow cytometry. The fluorescent intensity was partially correlated with H2O2 production by each neutrophil.
In case of carrier of chronic granulomatous disease there were two subpopulations of neutrophils, one was normal responding cells and the other was non-responding. Cells from septic patients revealed populations of neutrophils with increased oxidative responses.
