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. 2019 Nov 26;9:17610. doi: 10.1038/s41598-019-53855-2

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© The Author(s) 2019

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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In vitro nephrotoxicity assessment of kidney spheroids. (a) Live/dead stained images and apoptosis profile as analyzed by MUSE analyzer in 2D cultured cells (left) and mouse kidney spheroids (right) incubated with 20 μM cisplatin (CPT) or 50 μM cyclosporin A (CSA) for 24 h. Cell viability assay was performed by the Muse Count & Viability reagent (Millipore) following the manufacturer’s protocols. The results were obtained with Muse Count & Viability software module and the statistics are shown as the percentage of viable cells and annexin V-positive apoptotic cells. (b) Relative mRNA expression of kidney injury markers, such as Havcr1, Lcn2, clusterin, and caspase-3, in the kidney spheroids after treatment with nephrotoxicants. (c,d) Western blotting analysis (c) and immunostaining images (d) of kidney injury markers in kidney spheroids after treatment with 20 μM cisplatin (CPT) or 50 μM cisplatin cyclosporin A (CSA). All data are shown as the means ± s.e.m. *p < 0.05 compared with control by unpaired Student’s t-test. Scale bar, 20 μm.