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 concentration) were investigated and storage-related changes in erythrocyte response to oxidant stress explored. METHODS: Erythrocytes were incubated with a lipophilic fluorescent probe and exposed to site-specific oxidant challenge, induced by cumene hydroperoxide, in the presence and absence of urate, ascorbate, or alpha tocopherol in physiological amounts. Fluorescence of labeled and treated erythrocytes was measured for 120 min using a Coulter EPICS Elite ESP flow cytometer. RESULTS: Probe loading was dose and time dependent. Cumene hydroperoxide exhibited a potent and dose-dependent oxidant effect on erythrocyte membranes. Alpha tocopherol slowed, but did not prevent, membrane oxidation. Ascorbate appeared to have no effect on peroxidation initially, but then slowed and 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 slowing membrane peroxidation, but ascorbate appears to stop chain propagation. This effect may be owing to vitamin C/E interaction. Further study is needed.