Nienke Ruijter
Improving the dichloro-dihydro-fluorescein (DCFH) assay for the assessment of intracellular reactive oxygen species formation by nanomaterials
Ruijter, Nienke; van der Zee, Margriet; Katsumiti, Alberto; Boyles, Matthew; Cassee, Flemming R.; Braakhuis, Hedwig
Authors
Margriet van der Zee
Alberto Katsumiti
Dr Matthew Boyles M.Boyles2@napier.ac.uk
Lecturer
Flemming R. Cassee
Hedwig Braakhuis
Abstract
To facilitate Safe and Sustainable by Design (SSbD) strategies during the development of nanomaterials (NMs), quick and easy in vitro assays to test for hazard potential at an early stage of NM development are essential. The formation of reactive oxygen species (ROS) and the induction of oxidative stress are considered important mechanisms that can lead to NM toxicity. In vitro assays measuring oxidative stress are therefore commonly included in NM hazard assessment strategies. The fluorescence-based dichloro-dihydro-fluorescein (DCFH) assay for cellular oxidative stress is a simple and cost-effective assay, making it a good candidate assay for SSbD hazard testing strategies. It is however subject to several pitfalls and caveats. Here, we provide further optimizations to the assay using 5-(6)-Chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA-AE, referred to as DCFH probe), known for its improved cell retention.
We measured the release of metabolic products of the DCFH probe from cells to supernatant, direct reactions of CM-H2DCFDA-AE with positive controls, and compared the commonly used plate reader-based DCFH assay protocol with fluorescence microscopy and flow cytometry-based protocols. After loading cells with DCFH probe, translocation of several metabolic products of the DCFH probe to the supernatant was observed in multiple cell types. Translocated DCFH products are then able to react with test substances including positive controls. Our results also indicate that intracellularly oxidized fluorescent DCF is able to translocate from cells to the supernatant. In either way, this will lead to a fluorescent supernatant, making it difficult to discriminate between intra- and extra-cellular ROS production, risking misinterpretation of possible oxidative stress when measuring fluorescence on a plate reader.
The use of flow cytometry instead of plate reader-based measurements resolved these issues, and also improved assay sensitivity. Several optimizations of the flow cytometry-based DCFH ISO standard (ISO/TS 19006:2016) were suggested, including loading cells with DCFH probe before incubation with the test materials, and applying an appropriate gating strategy including live-death staining, which was not included in the ISO standard.
In conclusion, flow cytometry- and fluorescence microscopy-based read-outs are preferred over the classical plate reader-based read-out to assess the level of intracellular oxidative stress using the cellular DCFH assay.
Citation
Ruijter, N., van der Zee, M., Katsumiti, A., Boyles, M., Cassee, F. R., & Braakhuis, H. (2024). Improving the dichloro-dihydro-fluorescein (DCFH) assay for the assessment of intracellular reactive oxygen species formation by nanomaterials. NanoImpact, 35, Article 100521. https://doi.org/10.1016/j.impact.2024.100521
Journal Article Type | Article |
---|---|
Acceptance Date | Jun 18, 2024 |
Online Publication Date | Jun 17, 2024 |
Publication Date | 2024-07 |
Deposit Date | Jun 20, 2024 |
Publicly Available Date | Jun 20, 2024 |
Print ISSN | 2452-0748 |
Electronic ISSN | 2452-0748 |
Publisher | Elsevier |
Peer Reviewed | Peer Reviewed |
Volume | 35 |
Article Number | 100521 |
DOI | https://doi.org/10.1016/j.impact.2024.100521 |
Keywords | Dichloro-dihydro-fluorescein assay, DCFH assay, Reactive oxygen species, ROS, Oxidative stress, SSbD, Hazard screening |
Files
Improving the dichloro-dihydro-fluorescein (DCFH) assay for the assessment of intracellular reactive oxygen species formation by nanomaterials
(4 Mb)
PDF
Publisher Licence URL
http://creativecommons.org/licenses/by-nc-nd/4.0/
You might also like
Bayesian based similarity assessment of nanomaterials to inform grouping
(2022)
Journal Article
Downloadable Citations
About Edinburgh Napier Research Repository
Administrator e-mail: repository@napier.ac.uk
This application uses the following open-source libraries:
SheetJS Community Edition
Apache License Version 2.0 (http://www.apache.org/licenses/)
PDF.js
Apache License Version 2.0 (http://www.apache.org/licenses/)
Font Awesome
SIL OFL 1.1 (http://scripts.sil.org/OFL)
MIT License (http://opensource.org/licenses/mit-license.html)
CC BY 3.0 ( http://creativecommons.org/licenses/by/3.0/)
Powered by Worktribe © 2024
Advanced Search