Troubleshooting the dichlorofluorescein assay to avoid artifacts in measurement of toxicant-stimulated cellular production of reactive oxidant species.

Introduction: The dichlorofluorescein (DCF) assay is a popular method for measuring cellular reactive oxidant species (ROS). Although caveats have been reported with the DCF assay and other compounds, the potential for artifactual results due to cell-free interactions between the DCF compound and toxicants has hardly been explored. We evaluated the utility of the DCF assay for measuring ROS generation by the toxicants mono-(2-ethylhexyl) phthalate (MEHP), and tetrabromobisphenol A (TBBPA).

Methods: DCF fluorescence was measured spectrofluorometrically after a 1-h incubation of toxicants with 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA). MEHP was incubated with carboxy-H2DCFDA in cell-free solutions of Hank's buffered salt solution (HBSS), or in Royal Park Memorial Institute (RPMI) medium with or without fetal bovine serum. TBBPA was incubated with carboxy-H2DCFDA in cell-free HBSS and with human trophoblast cells (HTR8/SVneo cells).

Results: MEHP did not increase fluorescence in solutions of carboxy-H2DCFDA in HBSS or RPMI medium without serum. However, MEHP (90 and 180μM) increased DCF fluorescence in cell-free RPMI medium containing serum. Furthermore, serum-free and cell-free HBSS containing 25μM TBBPA exhibited concentration-dependent increased fluorescence with 5-100μM carboxy-H2DCFDA (p<0.05), but not 1μM carboxy-H2DCFDA. In addition, we observed increased fluorescence in HTR8/SVneo cell cultures exposed to TBBPA (0.5-25μM) (p<0.05), as we had observed in cell-free buffer.

Discussion: MEHP demonstrated an interaction with serum in cell-free generation of DCF fluorescence, whereas TBBPA facilitated conversion of carboxy-H2DCFDA to the fluorescent DCF moiety in the absence of serum. Because TBBPA increased fluorescence in the absence of cells, the increased DCF fluorescence observed with TBBPA in the presence of cells cannot be attributed to cellular ROS and may, instead, be the result of chemical activation of carboxy-H2DCFDA to the fluorescent DCF moiety. These data illustrate the importance of including cell-free controls when using the DCF assay to study toxicant-stimulated cellular production of ROS.