Cell viability of Hly A - exposed polymorphonuclear neutrophils (PMN) was assessed by propidium uptake, measured by flow cytometry. Hemolytic supernatant, but not the non hemolytic controls, caused a dose-dependent fluorescence signal in PMN. Cells exposed to low hemolytic activities (below 0,5 HU50/ml) did not fluoresce, although cell size, estimated by Forward Scatter (FSC), increased slightly, and then returned to normal within 30-60 minutes, suggesting both membrane damage in absence of propidium uptake and short term cell recovery from the effects of Hly A. The fluorescent signal from permeated PMN decreased 15 minutes after exposure to Hly A, a decrease which was prevented by chelation of extracellular Ca⁺² with EGTA. Whereas Ca⁺² entry into the cell is responsible for triggering mechanisms leading to loss of fluorescence, low or chelated extracellular Ca⁺² facilitate propidium uptake, but the fluorescent signal does not decrease only when both intracellular and extracellular Ca⁺²are chelated. The findings of this study, together with data from other authors, are taken as the basis to formulate a hypothethical sequence of events to explain the cytometric data obtained from Hly A exposed PMN, including the significance of increases in cell size without propidium uptake.

Polymorphonuclear leukocytes; alpha hemolysin; flow cytometry; fluorescent