Probe-assisted flow cytometric analysis of erythrocyte membrane response to site-specific oxidant stress

Background: Probe-assisted flow cytometry was used to monitor the response of membranes of living cells to oxidant stress in the presence and absence of antioxidants. Test conditions (fluorophore loading, oxidant concentratio n) were investigated and storage-related changes in erythrocyte response to oxidant stress explored. Methods: Erythrocytes were incubated with a lipophilic fluorescent probe an d exposed to site-specific oxidant challenge, induced by cumene hydroperoxi de, in the presence and absence of urate, ascorbate, or alpha tocopherol in physiological amounts. Fluorescence of labeled and treated erythrocytes wa s measured for 120 min using a Coulter EPICS Elite ESP flow cytometer. Results: Probe loading was dose and time dependent. Cumene hydroperoxide ex hibited a potent and dose-dependent oxidant effect on erythrocyte membranes . Alpha tocopherol slowed, but did not prevent, membrane oxidation. Ascorba te appeared to have no effect on peroxidation initially, but then slowed an d stopped propagation of membrane oxidation. The effect of urate was slight . Conclusions: This technique can provide insight into oxidative processes at the cellular level. Results indicated that lipophilic alpha tocopherol was the most effective antioxidant in slewing membrane peroxidation, but ascor bate appears to stop chain propagation. This effect may be owing to vitamin C/E interaction. Further study is needed. (C) 2000 Wiley-Liss, Inc.