The intracellular oxidation of 2 ',7 '-dichlorofluorescin in murine T lymphocytes

Intracellular reactive oxygen species (ROS) production by activated murine T lymphocytes was investigated by analyzing intracellular dichlorofluoresci n (DCFH2) oxidation in lymph node cells (LNC). An increase in DCFH2 oxidati on in LNC induced by phorbol myristate acetate (PMA) was detected by how cy tometry. It was confirmed that this increase was present in Thy1(+) LNC. We examined the contribution to intracellular DCFH2 oxidation of ROS released by leukocytes other than T cells present in the LNC suspension. Superoxide dismutase, catalase, and glutathione/glutathione peroxidase inhibited the PMA-induced increase in intracellular DCFH2 oxidation. Furthermore, PMA fai led to elicit DCFH2 oxidation in LNC isolated from mice lacking a functiona l NADPH oxidase (gp91(phox) gene knockout mice), but this response could be restored in these cells by the addition of T cell-depleted LNC from wild-t ype litter mates. This study highlights the necessity for caution in using the DCFH2 assay to demonstrate specific intracellular ROS production in het erogeneous cell populations. It also suggests that cells other than T cells in lymph node populations may, through production of ROS, influence the in tracellular redox state of T lymphocytes. (C) 2000 Elsevier Science Inc.