Cold atmospheric plasma (CAP) is a potential anticancer therapy. CAP has cytotoxic effects when applied either directly to cancer cell cultures or indirectly through plasma-conditioned liquids. This protocol describes how to treat adherent cultures of human cancer cell lines with CAP or plasma-conditioned medium and determine cell viability following treatment. The protocol also includes details on how to quantify the reactive oxygen and nitrogen species present in medium following CAP treatment, using chemical probes using UV-visible or fluorescence spectroscopy. CAP treatment takes ~30 min, and 3 h are required to complete quantification of reactive oxygen and nitrogen species. By providing a standardized protocol for evaluation of the effects of CAP and plasma-conditioned medium, we hope to facilitate the comparison and interpretation of results seen across different laboratories.
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This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 714793). Authors belong to the SGR2017 1165. M.P.G. and C.C. acknowledge the Generalitat de Catalunya for the ICREA Academia Award. Authors acknowledge the technical support of M. Sánchez.
The authors declare no competing interests.
Peer review information Nature Protocols thanks Sander Bekeschus, Hiromasa Tanaka and the other, anonymous reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Key references using this protocol
Tornin, J. et al. Sci. Rep. 9, 10681 (2019): https://doi.org/10.1038/s41598-019-47128-1
Mateu-Sanz, M. et al. Cancers 12, 227 (2020): https://doi.org/10.3390/cancers12010227
Tornin, J. et al. Free Rad. J Biol. Med. 164, 107 (2021): https://doi.org/10.1016/j.freeradbiomed.2020.12.437
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Tornin, J., Labay, C., Tampieri, F. et al. Evaluation of the effects of cold atmospheric plasma and plasma-treated liquids in cancer cell cultures. Nat Protoc 16, 2826–2850 (2021). https://doi.org/10.1038/s41596-021-00521-5