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. This protocol explains how to treat cancer cells in culture directly with cold atmospheric plasma or indirectly with plasma-conditioned medium. Quantification of the reactive oxygen species present following treatment is described. Upscaling of the treatments to allow molecular biology assays is also described.