SINGLE-CELL INVESTIGATION BY LASER-SCANNING CONFOCAL MICROSCOPY OF CYTOCHEMICAL ALTERATIONS RESULTING FROM EXTRACELLULAR OXIDANT CHALLENGE

Confocal laser scanning fluorescence microscopy coupled to image analy sis was employed in order to develop and evaluate procedures for the a ppraisal at the single-cell level of: (1) protein-bound 4-hydroxynonen al, the specific product of membrane peroxidation (by means of immunoc ytochemistry with biotin-avidin revelation); (2) protein oxidation (by reaction of protein carbonyls with 2,4-dinitrophenyl-hydrazine follow ed by immunocytochemistry of dinitrophenyl moieties); and (3) cellular protein thiols (by direct alkylation of sulfhydryl groups with thiol- specific fluorescent reagents possessing different cell permeabilities ). The procedures proved able to reveal the subcellular distribution o f cytochemical parameters useful as indices of oxidative stress condit ions, and may allow ''redox phenotyping'' of isolated cells, which wou ld provide an efficient tool in selected experimental models.