Novel use of the fluorescent dye 5-(and-6)-chloromethyl SNARF-1 acetate for the measurement of intracellular glutathione in leukemic cells and primary lymphocytes

Abstract

Glutathione (GSH) plays an important role in protecting cells against injury, particularly during oxidative stress. Alterations in GSH metabolism are becoming the focus of attention in many diseases such as cancer, neurodegeneration, and AIDS. As such, a rapid assessment of GSH levels in a clinical setting is of increasing importance. We tested the efficacy of the thiol-labeling fluorescent dye CM-SNARF in its ability to measure variations in GSH concentration using a visible-light flow cytometer. GSH levels in 183, Jurkat, and primary lymphocytes were depleted with buthionine sulfoximine (BSO) or diamide, or increased with N-acetylcysteine (NAC). Following each treatment, cells were divided and either labeled with CM-SNARF followed by flow cytometry analysis, or assayed for GSH using a biochemical method. BSO treatment caused a maximal 87-90% decrease in GSH and 68-76% decrease in fluorescence units. Diamide depleted GSH 91-95%, corresponding to a fluorescence decrease of 85-88%. NAC treatment increased GSH levels 27% and fluorescence 12-19%. The overall correlation (R2) between mean GSH concentration and mean fluorescence was 0.80-0.88. CM-SNARF can be used to semi-quantitatively and rapidly determine intracellular variations in GSH concentration in the range of 10-150 nmoles GSH/mg protein. © 2007 International Society for Analytical Cytology.