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. 2022 Jul 21;12:12491. doi: 10.1038/s41598-022-16776-1

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© The Author(s) 2022

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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The 29F.1A12 and RMP1-30 anti-PD-1 antibody clones recognize overlapping B16-F10 melanoma subpopulations. (a,b) Representative FACS plots of 29F.1A12 and RMP1-30 co-staining (left) and % dual positivity (mean ± s.e,m., calculated as a fraction of RMP1-30-reactive cells) of PD-1 surface protein expression (right) by live (FVD⁻) (a) B16-F10 wild-type (WT) versus PD-1-overexpressing (OE) melanoma cells or (b) unactivated versus activated WT T-cells (C57BL/6) reveals co-localization of PD-1 antibody binding. (c,d) Representative histograms (left) and % positivity (mean ± s.e.m.) of PD-1 surface protein expression (right) by live (FVD⁻) B16-F10 WT cells grown in standard (2D, black line) versus tumor spheroid (3D, red line) culture conditions, as determined by the (c) 29F.1A12 or (d) RMP1-30 anti-mouse PD-1 antibody clones. Results are representative of at least n = 3 independent experiments. *p < 0.01; ***p < 0.001.