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. 2020 Oct 28;10:18433. doi: 10.1038/s41598-020-74128-3

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© The Author(s) 2020

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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Immunological characterization of mixed populations of human SCs and fibroblasts. (a) Useful antibody combinations for co-immunostaining of fixed cells identifying SCs (left), fibroblasts (right) and both cell types (middle panels). Arrowheads in all panels point to selected fibroblasts characterized by the lack of p75^NGFR, Sox10, S100β and nestin. High levels of vimentin (intracellular, red) and CD44 (membrane, green) were present in SCs and fibroblasts. Most fibroblast cultures contained SMA positive (arrowheads) and negative (arrows) cells. (b–c) Cell surface immuno-labeling of live human SCs. (b) Comparison between O4 and p75^NGFR immuno-detection in adherent cells. Sub-confluent cultures of human SCs were incubated for 3 days in SC growth medium (+ mitogens) or in DMEM containing 1% FBS (- mitogens) prior to live cell labeling with O4 and p75^NGFR antibodies (green). Immunostaining with S100β antibodies (red) and DAPI (blue) was carried out after fixation. p75^NGFR levels were high and homogeneous both in the absence and in the presence of mitogenic factors (b, lower panels). By contrast, O4 levels were variable and mitogen-dependent (b, upper panels). Some S100β positive SCs did not express O4 (b, white arrows) despite prolonged incubation in SC growth medium. (c) Fluorescence microscopy imaging (upper panels) and flow cytometry analysis (lower panels) of human cultures affected by various degrees of fibroblast contamination (indicated as high and low in the upper panels). p75^NGFR antibodies were used to stain adherent cells (fluorescence microscopy) and cells in suspension (flow cytometry). Thy-1 antibodies (CD90) were used only for flow cytometry analysis due to empirical data showing ineffective detection in adherent (live or fixed) cells. These images attest to the high specificity of S100β (cytoplasmic) and p75^NGFR (membrane) co-immunodetection in SCs but not fibroblasts. Whereas Thy-1 (CD90) expression was absent in SCs (c), the levels of Thy-1 were heterogeneous in p75^NGFR negative cells.