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. 2017 Nov 9;7:15130. doi: 10.1038/s41598-017-15417-2

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© The Author(s) 2017

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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Flow cytometry analysis of nanobody cell binding. The cell binding of the 4hD29 nanobody (Nb) was evaluated by flow cytometry. (A–D) 4hD29 (red) recognizes human DPP6 in different cell types (HsDPP6); (A) 4hD29 (red) labelled transiently-transfected CHO cells overexpressing DPP6, where neither the irrelevant control Nb (green) nor the secondary control antibodies only (blue) stained the cells (n = 3); (B) non-transfected CHO cells labelled as in A (n = 3); (C) The Nb 4hD29 (red) recognizes DPP6-positive human Kelly neuroblastoma (n = 4) and (D) EndoC-βH1 cells (n = 5), whereas the secondary antibodies only (blue) or an irrelevant control Nb (green) do not. (E,F) 4hD29 (red) binds to endocrine (TSQ⁺/Rh⁻) (E), but not to exocrine tissue (TSQ⁻/Rh⁺) (F) of dissociated human pancreas (n = 4). Background staining with secondary staining control is indicated in blue. (G) Overview of the gating strategy for endocrine (TSQ⁺/Rh⁻) and exocrine cells (TSQ⁻/Rh⁺) analysed in (E,F). The median fluorescence intensity (MFI) was calculated for Kelly neuroblastoma (H) and EndoC-βH1 cells (I). Delta MFI values were calculated to compare the endocrine (TSQ⁺/Rh⁻) and exocrine populations (TSQ⁻/Rh⁺), showing that 4hD29 has an increased binding in endocrine cells as compared to exocrine cells (J). Unpaired (E,F) or paired (J) Student’s t-tests were performed to compare two groups; *p ≤ 0.05, **p ≤ 0.01.