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. 2021 Mar 19;19(3):163. doi: 10.3390/md19030163

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© 2021 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Determination of ROS levels at the single cell level by flow cytometry using the fluorescent probe DCF-DA.(a) Experiments carried out with cells treated with empty microparticles (P). Overall, the reported differences are statistically significant ( $F_{4, 20} = 284.5$ , $p < 2 \times 10^{- 16}$ , ANOVA test). H $_{2}$ O $_{2}$ significantly increases intracellular ROS with respect to controls, but ROS levels in cells treated with H $_{2}$ O $_{2}$ and empty particles were not statistically different from cells treated with H $_{2}$ O $_{2}$ alone, suggesting that, within the power of the measurement, empty particles cannot reverse intracellular H $_{2}$ O $_{2}$ -induced ROS accumulation ( $p = 0.85$ , Tukey-HSD post-hoc test). (b) Experiments carried out with ASX-loaded microparticles (P-ASX). The reported differences are statistically significant ( $F_{4, 20} = 5828$ , $p < 2 \times 10^{- 16}$ , ANOVA test). ASX-loaded microparticles can significantly reduce intracellular H $_{2}$ O $_{2}$ -induced ROS accumulation ( $p < 10^{- 8}$ , Tukey-HSD post-hoc test). In both panels, the labels refer to: ctrl, cell autofluorescence; probe, fluorescence measured with cells loaded with the DCF-DA probe; P or P-ASX, fluorescence measured from cells loaded with DCF-DA and treated with empty (P) or ASX-loaded (P-ASX) microparticles; H $_{2}$ O $_{2}$ , fluorescence measured from cells loaded with DCF-DA and treated with H $_{2}$ O $_{2}$ ; P or P-ASX + H $_{2}$ O $_{2}$ , fluorescence measured from cells loaded with DCF-DA, treated with H $_{2}$ O $_{2}$ and with empty (P) or ASX-loaded (P-ASX) microparticles.