A method to determine reactive oxygen species production in intestinal and liver cell cultures using the 2 ′ ,7 ′ -dichlorodihydrofluorescein diacetate assay
Date
2024-02-13
Authors
Engelbrecht, Ilzé
Horn, Suranie
Giesy, John
Pieters, Rialet
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Elsevier
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Article
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Abstract
Exposure to xenobiotics can increase the production of reactive oxygen species (ROS). When detoxification organs such as the intestines and liver cannot neutralise these xenobiotics, it can induce oxidative stress and cause damage to tissues. Therefore, cell-based bioassays that indicate intracellular ROS production are a useful screening tool to evaluate the effect of these chemicals. Although flow cytometry is commonly used to measure ROS in cells, many research laboratories in the Global South do not always have access to such specialised instrumentation. Therefore, we describe a sensitive but low-cost method that can easily be used to determine ROS production in vitro. This method employs the fluorogenic dye, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA), which emits fluorescence after being oxidised to a fluorescent derivative. Since the H2DCF-DA bioassay indicates non-specific ROS production it can be used as a marker of overall oxidative stress. This method was validated by exposing human duodenum epithelial adenocarcinoma (HuTu-80) and rat liver epithelial hepatoma (H4IIE-luc) cells to agricultural soil samples.
Description
The version of record of this article, first published in MethodsX, is available online at Publisher’s website: https://doi.org/10.1016/j.mex.2024.102615
Keywords
96-well microplate, Mammalian cell lines, Fluorescence, Fluorogenic probe, In vitro bioassay, Oxidative stress
Citation
Engelbrecht, I.S. Horn, J.P. Giesy, R. Pieters. 2024. A method to determine reactive oxygen species production in intestinal and liver cell cultures using the 2’ 7’dichlorodihydrofluorescein diacetate assay. Methods X. 12:102615
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DOI
10.1016/j.mex.2024.102615