Refinement of the dichlorofluorescein assay for flow cytometric measurement of reactive oxygen species in irradiated and bystander cell populations.
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) gamma-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 gamma 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.
Hafer
Kurt
K
Department of Radiation Oncology, David Geffen School of Medicine at UCLA, Los Angeles, California 90095, USA.
Iwamoto
Keisuke S
KS
Schiestl
Robert H
RH
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D024201:Bystander Effect; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004307:Dose-Response Relationship, Radiation; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000032:analysis* / Q000528:radiation effects*
D005452:Fluoresceins; D017382:Reactive Oxygen Species; C037631:2',7'-dichlorofluorescein; D001205:Ascorbic Acid; D000111:Acetylcysteine