Refinement of the Dichlorofluorescein Assay for Flow Cytometric Measurement of Reactive Oxygen Species in Irradiated and Bystander Cell Populations

• Published in: Radiation research 2008-04, Vol.169 (4), p.460-468
• Main Authors: Hafer, Kurt, Iwamoto, Keisuke S., Schiestl, Robert H.
• Format: Article
• Language: English
• Subjects: Acetylcysteine - pharmacology; Animals; Antioxidants; Ascorbic Acid - pharmacology; Bystander Effect; CHO Cells; Cricetinae; Cricetulus; Cultured cells; Cytometry; Dose response relationship; Dose-Response Relationship, Radiation; Flow Cytometry - methods; Fluoresceins - metabolism; Fluorescence; Irradiation; Oxidative stress; Radiation dosage; Reactive oxygen species; Reactive Oxygen Species - analysis; Reactive Oxygen Species - radiation effects; Technical Advance
• ISSN: 0033-7587; 1938-5404
• DOI: 10.1667/RR1212.1

Hafer, K., Iwamoto, K. S. and Schiestl, R. H. Refinement of the Dichlorofluorescein Assay for Flow Cytometric Measurement of Reactive Oxygen Species in Irradiated and Bystander Cell Populations. Radiat. Res. 169, 460–468 (2008). Reactive oxygen species (ROS) have been implicated in many ionizing radiation-related phenomena, including bystander effects. The oxidation of 2′7′-dichlorofluorescin (DCFH) to fluorescent 2′7′-dichlorofluorescein (DCF) is commonly used for the detection of radiation-induced ROS. The DCF assay was adapted for efficient, systematic flow cytometry quantification of low-linear energy transfer (LET) γ-radiation-induced ROS in vitro in Chinese hamster ovary (CHO) cells. This method is optimized for increased sensitivity to radiation-induced ROS and to discriminate against measurement of extracellular ROS. This method can detect a significant increase in ROS in cells exposed to γ radiation at doses as low as 10 cGy. The antioxidants N-acetyl-cysteine and ascorbic acid (vitamin C) significantly reduced the amount of ROS measured in cells exposed to 5 Gy ionizing radiation. This method was used to measure the intracellular ROS in unirradiated CHO bystander cells co-cultured with low-LET-irradiated cells. No increase in ROS was measured in bystander cell populations co-cultured with the irradiated cells beginning 9 s after radiation exposure.