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. 2017 May 31;8:15648. doi: 10.1038/ncomms15648

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Copyright © 2017, The Author(s)

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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(a–e) Splenic pDCs were plated on coverslips with or without high-dose gp96, and in the presence or absence of Nrp1 blocking antibody (α-Nrp1) or isotype control antibody (Iso). Before imaging, Treg (CD4⁺CD25⁺⁾ or Tconv (CD4⁺CD25⁻) were labelled with Cell Tracker Red dye and added to the pDC culture dishes at a 2:1 DC:T-cell ratio. Images were acquired at 5 min intervals for a total of 1–2 h. Interaction and motility were analysed. (b) Treg were tracked using NIS Elements tracking software. Representative Treg-pDC interactions are shown for each treatment group for the indicated times. The interaction times of Tregs with pDCs from multiple images are quantified. All Tregs (including those that failed to interact with pDC) are included. (c) The average percentage of Tregs which undergo long (>10 min) or short to no (0–10 min) interactions with pDCs were calculated for each group. (d,e) Tconv were analysed similarly to Treg. Total interaction time (d) and percentage of long interaction (e) are shown. All data are from one representative experiment of 2–3 independent experiments. Data are represented as mean±s.d. ns, not significant, *P<0.05, **P<0.01 (one-way ANOVA).