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. 2014 Jan 2;4:501. doi: 10.3389/fimmu.2013.00501

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Copyright © 2014 Petvises and O’Neill.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Investigation of pre-cDC production in co-cultures. The presence of pre-cDC was investigated in (A) cells collected from 14-day co-cultures of C57BL/6J Lin⁻ BM over 5G3 stroma, and in (B) C57BL/6J BM. Progenitors were gated as a Lin⁻ subset using FSC and SSC plots. Pre-cDC were detected by staining for c-kit, Sca-1, Flt3, CD11c, CD45RB, CD43, and Sirpα. Propidium iodide (PI) was added to allow gating of PI⁻ live cells. Gates were set on bivariate plots using isotype control antibodies, and numbers on gates reflect % positive cells. Using the gating strategy shown in (B), pre-cDC were sorted out of BM for co-culture above 5G3 stroma. (C) Production of dendritic-like cells in pre-cDC co-cultures was assessed in relation to Lin⁻ BM co-cultures after 14 days following gating of large cells on a bivariate CD11c versus CD11b plot, and analysis of expression of CD11c, CD11b, MHC-II, and CD8α.