Fig 1: Photomicrographs of CTCs isolated from LARC patients, stained with anti-PD-L1 (A), anti-RAD-23B (B), anti-TGF-ßRI (C) and anti-TYMS (D). In (E), a CTC with hematoxylin, without any antibody staining. Following, in boxes (F–H), positive controls (cell lineages, that accordingly to Protein Atlas (https://www.proteinatlas.org/ accessed on 13 December 2016) express the studied antibodies, spiked in healthy individual blood). In (F), FADU lineage, expressing PD-L1. In box (G), HCT-8 cells expressing RAD23B. In (H), A549 cells with TGF-ßRI staining; in (I,J), leucocytes staining for TYMS and CD45, respectively. In boxes (K,M,N), negative controls (cell lineages, that accordingly to Protein Atlas (https://www.proteinatlas.org/ accessed on 13 December 2016) do not express or express very low levels of the studied antibodies, spiked in healthy individual blood). In (K), PC3 lineage without any expression of PD-L1. In (M), MCF7 without TGF-ßRI staining and in (N), U87MG without TYMS expression. In (L), leucocytes from healthy individuals without RAD23B staining. For negative control of CD45, we used A549 lineage.
Fig 2: Stromal cell isolation based on data-driven gating strategies distinguishes cell subsets with different colony-forming capacities and differentiation capacities.(A) Dot plot of surface marker gene expression in different stromal cell clusters. Cluster numbers and corresponding stromal cell groups are indicated on the y-axis legend. Dot sizes represent the percentage of cells expressing a certain gene in each cluster and dot colors represent the scaled average expression of that gene. (B) FACS plots illustrating the gating strategy for the isolation of different stromal subsets. The displayed cell populations are indicated on top of the plot. Following exclusion of doublets, dead cells, CD45- and CD235a-expressing cells, CD45low/-CD235a-CD71-CD271+ cells (left panel) were gated based on CD52 and NCAM1 expression (middle panel). The resulting three populations were labelled as A-C (middle panel), corresponding to the stromal cell groups in (A) (A, CD52-NCAM1-; B, CD52-NCAM1+; C, CD52+NCAM1-). CD45-CD235a-CD71-CD271+CD52-NCAM1- (group A) cells were further divided based on CD81 expression and four populations were identified (A1–A4) (right panel). A1, CD81++; A2, CD81+; A3; CD81+/-; A4, CD81-. (C) CFU-F frequencies of sorted stromal cell populations as shown in (B). Data are presented as individual data (dots) and median (horizontal lines) from independent experiments (n=3–6). Symbol colors and x-axis labels correspond to the cell population colors in (B). *: p<0.05; ****: p<0.0001 (Kruskal-Wallis test). (D) In vitro differentiation capacity of sorted stromal cell populations (as indicated in B) towards the adipogenic, osteoblastic, and chondrogenic lineage. Non-induction controls are shown in the left panel. Scale bars represent 200 µm. A representative set of pictures from a total of three independent experiments is shown. (F) Formalin-fixed, paraffin-embedded (FFPE) human BM slides were sequentially stained for DAPI (blue), CD45 (yellow), CD81 (green), CD271 (pink), and NCAM1 (cyan) and scanned with the OlympusVS120 slide scanner. Different staining combinations are shown as indicated under each picture to provide better visualization of individual staining obtained from the same FFPE slide. Scale bars represent 50 µm. Red arrows: CD271+CD81++ cells; white arrows: arteriolar walls; white lines: bone lining regions. Bone (b), adipocytes (a), and capillaries (*) are indicated.
Fig 3: Expression of selected ligand and receptor pairs involved in cell-cell interaction between stromal cells and hematopoietic cells in human bone marrow.(A–D, F–G) UMAP (as in Figure 1B) illustration of the normalized expression of selected ligand and receptor gene pairs. Red dashed lines in figures A, B and D mark the erythroid clusters. The blow-up shown in (G) is to better visualize PDGFA- and PDGFB-expressing cells. (E) Left panel: Formalin-fixed, paraffin-embedded (FFPE) human BM slides were sequentially stained for DAPI (blue), CD45 (yellow), SPP1 (red), CD271 (pink), and NCAM1 (cyan) and scanned with an OlympusVS120 slide scanner. Left upper image: all markers are shown; left lower image: just NCAM1 channel is shown. Middle panel: confocal laser scanning analysis of BM biopsies co-stained with CD271 (red), NCAM1 (green), SPP1 (white), and DAPI (blue) presented as 3D orthographic cross-section view. Right panel: Single staining channel data for CD271 (red), NCAM1 (green), SPP1 (white), and corresponding blow-ups for indicated areas. NCAM1, SPP1 and CD271(NGFR). White scale bars represent 50 µm and yellow scale bars represent 25 µm. White dashed lines indicate the trabecular bone surface lining regions.
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