FLUORESCEINATED PHOSPHOETHANOLAMINE FOR FLOW-CYTOMETRIC MEASUREMENT OF LIPID-PEROXIDATION

A new lipophilic fluorescein probe (fluor-DHPE) has been identified th at can assay lipid peroxidation in mammalian cells on a cell-by-cell o r selected-cell-subpopulation basis by flow cytometry. Application of this approach requires that the fluorescent probe be nonexchangeable a mong cells. Fluorescein is an appropriate fluorophore, since its fluor escence matches the specifications of common flow cytometers and the c ompound loses its fluorescence upon reaction with peroxyl radicals. Up on examination of four lipophilic derivatives of fluorescein, fluor-DH PE was found to be the only probe that was nonexchangeable among label ed and unlabeled rat RBC for at least 24 h. The exposure of fluor-DHPE -labeled RBC to benzoyl peroxide followed by mixing the sample with RB C unexposed to peroxide led to a decrease in fluorescence. Furthermore , the flow cytometer could clearly select the subpopulation of cells u ndergoing lipid peroxidation from those cells that were not. Fluor-DHP E-labeled-RBC obtained from rats and expos ed to cumene hydroperoxide also displayed a gradual decrease in fluorescence. This decrease was p reventable by either regulation of the vitamin E content in the animal diet or in vitro supplementation of cells with vitamin E. We conclude that fluor-DHPE is a stable and nonexchangeable probe for monitoring lipid peroxidation in cell subpopulations by flow cytometry. (C) 1998 Elsevier Science Inc.