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. 2021 Feb 26;11:4735. doi: 10.1038/s41598-021-84023-0

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© The Author(s) 2021

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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Cell sorting strategy, phenotypic and functional characterization of CpG-induced c-kit⁺Sca-1⁺B220^intPDCA-1⁺ BM cells. (a) Total BM cells incubated with PBS or CpG-B (1 µg/ml) for 18 h and magnetically enriched for c-kit⁺ cells were further labelled for Sca-1, B220, PDCA-1 and electronically sorted into c-kit⁺Sca-1⁺B220^intPDCA-1⁺ cells. Flow cytometry dot plots representative of 30 experiments. (b) Further characterization of cell-sorted CpG- as well as PBS-induced BM population was performed using flow cytometry analysis of the expression of various myeloid and dendritic cell markers as well as cell migration receptors. Specific antibody staining is depicted with open histograms (blue line for PBS- and red line for CpG-induced cells). Positive cells are defined using FMO controls (grey histograms). Two experiments. (c) FACS analysis of intranuclear expression of transcription factors. Analysis of IRF8 and Id2 expression with specific antibodies compared to FMO controls and of E2.2 and PU.1 by flowRNA, compared with B220⁺PDCA-1⁻ control B cells (grey histograms), in CpG- versus PBS-stimulated c-kit⁺Sca-1⁺B220^intPDCA-1⁺ population. Two experiments. (d) Frequency of c-kit⁺Sca-1⁺B220^intPDCA-1⁺ cells emerging among total BM cells after 18 h of incubation with different TLR agonists, measured in BM cell cultures of four individual mice. Statistical significance as indicated, analyzed by Mann–Whitney test. (e) Cytokine production of cell-sorted PBS- versus CpG-induced c-kit⁺Sca-1⁺B220^intPDCA-1⁺ cells, measured in the supernatant after 4 h of activation with PMA/ionomycin in the presence of brefeldin A using multiplex ELISA. Biological duplicates. (f) Intracytoplasmic TGF-β expression was analyzed by FACS in PBS- and CpG-pre-pDCs. One experiment out of two (g) IFN-α production was measured by ELISA in supernatants of CpG- versus PBS-induced cell-sorted c-kit⁺Sca-1⁺B220^intPDCA-1⁺ BM cells after 18 h incubation with 1 μg/ml TLR-7 (R848), TLR-9 (CpG-B and CpG-P) and TLR-4 (LPS) agonists (biological duplicates). (h) Mature spleen pDCs and CpG-pre-pDCs were compared for their IFN-α response after 18-h incubation with CpG-B (1 μg/ml), measured by ELISA in supernatants (biological duplicates).