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. 2020 Nov 24;10:20440. doi: 10.1038/s41598-020-77066-2

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Measurement of intracellular ROS generation in L. donovani promastigotes. ROS generation was monitored by flow cytometry using H₂DCFDA at 3 h, 6 h, 12 h and 24 h. Elevated ROS level in β-sitosterol_CCL-treated parasites was observed compared to the control, whereas ROS generation in NAC-pre-treated promastigotes was found to be restrained to the level in control cells at each time point. Comparison of ROS generation in untreated, β-sitosterol_CCL (IC₅₀ dose)-treated and NAC (20 mM)-pre-treated promastigotes at different time points are represented in both the bar graph and histogram. (A) The bar graph is representative of three independent experiments, and statistical significance is calculated compared to the untreated control set by using one-way ANOVA with Dunnett's multiple comparison test, where *p < 0.05, **p < 0.01 and ****p < 0.0001 are considered statistically significant. (B) Histograms depict the shift of MFI as indicated by the H-1 region. Data were acquired in a BD FACSCalibur flow cytometer and analysed in Flowing software (https://www.flowingsoftware.com), version 2.5.1, Finland.