Detection of Reactive Oxygen Species Induced by Radiation in Cells Using the Dichlorofluorescein Assay

Abstract

The goal of this study was to determine the amount of reactive oxygen species (ROS) that arises inside cells irradiated in medium containing blood serum using the 2'7'-dichlorofluorescein (DCF) assay. DCF fluorescence in cells and medium was recorded on an MF44 Perkin Elmer fluorimeter, and fluorescence in cells only was recorded on a Partec flow-through cytometer. Human larynx tumor HEp-2 cells and lympholeukosis P388 cells were irradiated with X rays at a dose rate of 1.12 Gy/min. The factors (temperature, pH, serum concentration) affecting the oxidation of 2'7'-dichlorofluorescin (DCFH) to DCF were studied, and errors in the dichlorofluorescein assay of ROS were minimized. The amount of ROS registered by the DCF assay in cells was found to depend on the concentration of serum in the medium during irradiation. In the presence of 10% serum, radiation had no effect on the amount of detectable ROS. The effect of radiation on the formation of intracellular ROS was almost completely abolished if the irradiated medium was removed immediately after radiation exposure. The increase in the formation of ROS in cells irradiated in medium with a low serum content is due mainly to the radiolytic products of water that arise in medium and oxidize DCFH located in cells.

Role of H2O2 in RET/PTC1 Chromosomal Rearrangement Produced by Ionizing Radiation in Human Thyroid Cells

“…This result was in disagreement with the recent report of very low H 2 O 2 concentrations after irradiation of the cell medium by similar γ-ray doses and of the absence of a potentiating effect of L-buthionine-sulphoximine on DNA damage, a drug that decreases intracellular glutathione, and consequently, induces a protective effect for H 2 O 2 against degradation (13). This discrepancy could be due to our experimental conditions, because serum was withdrawn from the cell medium before irradiation to avoid H 2 O 2 produced during radiation exposure to be degraded by the catalase activity of the serum (30). It was previously observed that extracellular H 2 O 2 rapidly disappeared (13,31).…”

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

“…DCFH-DA can be photo-oxidized by UV and visible light (36); thus incubation, irradiation and FACS measurement should be done in the dark. ROS produced by normal oxidative metabolism can produce artifacts if DCF fluorescence is measured when the times after exposure are significantly different; such artifacts can be minimized if samples are kept on ice after exposure and are assayed at as nearly at the same time as possible (37). DCFH-DA present in the medium can also be oxidized extracellularly to DCF-DA after irradiation and then enter the cell to become fluorescent after deacetylation to DCF (see Fig.…”

“…A recent report (37) was highly critical of studies using DCFH-DA for quantifying intracellular radicals formed by radiation, especially the studies of Wan et al (20,28,32,34), and proposed that the DCF method is subject to an artifact due to an influx of extracellularly generated hydrogen peroxides after irradiation. We subsequently sought to measure the contribution of this artifact hypothesized by Korystov et al (37) and found that its contribution to DCF fluorescence in CHO cells is negligible (40).…”

Lupulone, a hop bitter acid, activates different death pathways involving apoptotic TRAIL-receptors, in human colon tumor cells and in their derived metastatic cells

“…Therefore we measured ROS production by flow cytometry using the fluorescent probe CM-H 2 DCFDA [31]. Green fluorescence generated by the reaction between the fluorescent probe and ROS was analysed in both SW480 and SW620 cells.…”