Redox Buffering Ability of Lymphoid Cells Evaluated by the Oxidation of 2′,7′-Dichlorofluorescin

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Abstract
The redox buffering activity of several lymphoid cells against endogenous and exogenous H2O2 has been evaluated using 2′,7′-dichlorofluorescin diacetate (DCFH2-DA). The mechanism of 2′,7′-dichlorofluorescin (DCFH2) oxidation has also been investigated. It was found that while the oxidation by external H2O2 is completely inhibited by azide or cyanide, the oxidation by endogenous species is still present, even under anaerobic conditions. The data herein reported indicate that autoxidation and peroxidation of DCFH2 are distinct reactions. Hence only by addition of increasing concentrations of exogenous hydrogen peroxide, the fluorescence of DCF can be used to evaluate the cellular ability of scavenging H202. By this method we have found that the erythroleukaemia cell line K56 and promyelocytic line HL-60 show a faster rate of DCFH2 oxidation than peripheral blood leukocytes (PBL), mature T-cells (MOLT-3 and MOLT-4) and B-cells (DAUDI). Using this method the balance between antioxidant enzymes activity and the redox state of the cell can be easily assessed by fluorescence both in single cells and in cell populations.