Chemically de-acetylated 2′,7′-dichlorodihydrofluorescein diacetate as a probe of respiratory burst activity in mononuclear phagocytes

Abstract

2′,7′-dichlorodihydrofluorescein diacetate (H 2DCFDA) is a fluorogenic probe commonly used to detect cellular production of reactive oxygen species (ROS), for example in the respiratory burst of granulocytes and mononuclear phagocytes. This method depends on the de-acetylation of H 2DCFDA by cellular esterases, to form the oxidant-sensitive compound, 2′,7′-dichlorodihydrofluorescein (H 2DCF). Importantly, however, not all cells possess sufficient esterase activity to produce the H 2DCF needed for accurate measurement of ROS. In this study, we used chemically de-acetylated probe (H 2DCF) to assess the phorbol-ester-triggered respiratory burst of rainbow trout macrophages, which, like some mammalian mononuclear phagocytes, appear to have low probe-esterase activity. We compared this approach to the use of intact H 2DCFDA and the cytochrome c reduction assay. The H 2DCF and cytochrome c reduction assays gave similar portrayals of the kinetics of the macrophage respiratory burst, while H 2DCFDA did not. We therefore recommend the use of H 2DCF over H 2DCFDA for quantification of the production of reactive oxygen species. Additionally, we stress the need to test reaction buffers or culture media used with H 2DCF(DA) for their ability to oxidize the probe directly or indirectly. As an example, we have observed that tyrosine combined with ubiquitous metal contaminants of physiological buffers can result in high levels of oxidation, which may be incorrectly interpreted as cellular activity.