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

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• 作者：Brubacher ; John L. ; Bols ; Niels C.
• 关键词：2&prime ; 7&prime ; -Dichlorodihydrofluorescein diacetate ; Respiratory burst ; Reactive oxygen species ; Macrophage ; Rainbow trout ; Tyrosine ; ANOVA ; Analysis of variance ; DCF ; 2&prime ; 7&prime ; -Dichlorofluorescein ; H₂DCF ; 2&prime ; 7&prime ; -Dichlorod
• 刊名：Journal of Immunological Methods
• 出版年：2001
• 期刊代码：57_00221759
• 类别：imm
• 出版时间：May 1, 2001
• 卷：251
• 期：1-2
• 页码：81-91
• 文件大小：291 K

摘要

2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) 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 H₂DCFDA by cellular esterases, to form the oxidant-sensitive compound, 2′,7′-dichlorodihydrofluorescein (H₂DCF). Importantly, however, not all cells possess sufficient esterase activity to produce the H₂DCF needed for accurate measurement of ROS. In this study, we used chemically de-acetylated probe (H₂DCF) 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 H₂DCFDA and the cytochrome c reduction assay. The H₂DCF and cytochrome c reduction assays gave similar portrayals of the kinetics of the macrophage respiratory burst, while H₂DCFDA did not. We therefore recommend the use of H₂DCF over H₂DCFDA for quantification of the production of reactive oxygen species. Additionally, we stress the need to test reaction buffers or culture media used with H₂DCF(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.