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Evaluation of the effects of cold atmospheric plasma and plasma-treated liquids in cancer cell cultures


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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Fig. 1: Procedure overview.
Fig. 2: Direct and indirect treatment can be performed in the same experimental setting.
Fig. 3: Chemical reactions used to detect RONS described in this protocol.
Fig. 4: Overview of how to use different sizes of dish for CAP and PCM treatments.
Fig. 5: Experimental steps to generate a calibration curve for detection of H2O2.
Fig. 6: Experimental steps to generate a calibration curve for detection of nitrites.
Fig. 7: Experimental steps to generate a calibration curve for detection of nitrates.
Fig. 8: Cell cytotoxicity assay applied to different dish sizes.
Fig. 9: Typical results following treatments in 24-well plates.
Fig. 10: Analysis of components known to be involved in cell signaling.

Data availability

Source data are available for Figs. 810 at


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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.

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Authors and Affiliations



J.T. and C.L. contributed to the acquisition, analysis and interpretation of data, optimization of the protocol and writing of the work. F.T. contributed to acquisition and analysis of data, and to revising it. M.P.G. contributed to writing, discussing and revising the work. C.C. conceived the work, contributed to writing and substantively revising it.

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Correspondence to Cristina Canal.

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The authors declare no competing interests.

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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.

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Key references using this protocol

Tornin, J. et al. Sci. Rep. 9, 10681 (2019):

Mateu-Sanz, M. et al. Cancers 12, 227 (2020):

Tornin, J. et al. Free Rad. J Biol. Med. 164, 107 (2021):

Supplementary information

Supplementary Information

Supplementary Table 1 and Supplementary Methods 1 and 2.

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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).

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