Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Comparison of Various Niches for Endothelial Progenitor Cell Therapy on Ischemic Myocardial Repair

doi: 10.1161/atvbaha.111.244970

Figure Lengend Snippet: Figure 3. Analysis of cell marker expression and functional characterization of the progenitor cells. A, Representative photomicrograph of the endothelial progenitor cell (EPC) colonies (a, magnification 100) characterized by typical cobblestone shape (d, magnification 400). Immunomicroscopy showed that most of EPCs labeled with Hoechst 33342 postinduction were double-stained with factor VIII, but not with myosin heavy chain (MHC). Factor VIII was found in large secretory vesicles (arrows). The nuclei of EPCs were stained blue with Hoechst 33342 (b and c). EPC cytoplasm stained red (arrows) with anti-factor VIII (e) or MHC (f) antibodies. DAPI indicates 4=,6-diamidino-2-phenylindole. B, Phenotype of gated EPCs evaluated by fluorescence-activated cell sorting. FITC indicates fluorescein isothiocyanate. C, Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiopoietin-1 (Ang-1), interleukin-1 (IL-1), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor (TNF) immunostaining in EPCs. The nuclei of EPCs stained blue with Hoechst 33342. EPC cytoplasm stained red (arrows) with anti-VEGF, bFGF, Ang-1, IL-1, MCP-1, or TNF antibodies. The high magnification shown in C is 400.

Article Snippet: In order to analyze the cell surface markers of EPCs, the following monoclonal antibodies (mAbs) conjugated to fluorochromes were used: anti-CD34-PE (MA1-19770; Thermo Scientific, Waltham, MA, USA), CD45-FITC (C2399-07L; United States Biological, Swampscott, MA, USA), CD14-FITC (20-783-74842; GenWay Biotech, San Diego, CA, USA), CD11b-PE (301307; BioLegend, San Diego, CA, USA), VEGFR2 (KDR, ab9530; Abcam, Cambridge, UK), and CD133 (AP2010b; Abgent, San Diego, CA, USA), For direct labeling, cells were incubated with 1 μg/ml of antibody for 30 min at room temperature.

Techniques: Marker, Expressing, Functional Assay, Labeling, Staining, FACS, Immunostaining

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Single-cell RNA sequencing reveals that an imbalance in monocyte subsets rather than changes in gene expression patterns is a feature of postmenopausal osteoporosis.

doi: 10.1093/jbmr/zjae065

Figure Lengend Snippet: Figure 1. Changes in clusters of bone marrow CD14+ monocytes in women with PMOP. (A) The distribution of bone marrow CD14+ monocytes from postmenopausal women with normal bone mass in UMAP was highly similar to that from women with PMOP. (B and C) Results for Monocle2 analysis. (D) The pseudotime analysis results were mapped to UMAP; the lighter the color is, the more primitive the cell is. (E) Volcano plot of the marker genes in each cluster. The marker genes are provided as a “cluster.markers” file. (F) Expression levels of CD14 mRNA in each cluster. (G) Expression levels of FCGR3A (CD16) mRNA in each cluster. (H) Average expression of CD14 and CD16 mRNAs in each cluster. (I) When the clusters were mapped to UMAP, the mapping positions of different clusters were similar, but there were differences in the proportion of clusters. (J) The stacked column chart of the proportions of 7 clusters in each sample. (K) The proportion of clusters 1 and 7 between healthy and PMOP samples. (L) The characteristic genes of each group were analyzed via GSEA.

Article Snippet: No antibody was added to the first tube, 5 μL of CD14 antibody (Proteintech, FITC-65056) was added to the second tube, 5 μL of CD16 antibody (Proteintech, PE65090) was added to the third tube, and 5 μL of CD14 antibody and 5 μL of CD16 antibody were added to the fourth tube.

Techniques: Marker, Expressing

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Single-cell RNA sequencing reveals that an imbalance in monocyte subsets rather than changes in gene expression patterns is a feature of postmenopausal osteoporosis.

doi: 10.1093/jbmr/zjae065

Figure Lengend Snippet: Figure 2. Developmental trajectory of bone marrow CD14+ monocytes. (A) Using the TSCAN package to perform pseudotime analysis, the smaller the abscissa value is, the more primitive the cell is. (B) Unsupervised inference of developmental directions for monocytes using VECTOR. (C) Map of the RNA velocity on UMAP. (D) Changes in dynamic expression of CD14 and the top 10 upregulated characteristic genes in cluster 1 and cluster 7 during differentiation of cluster 1 into cluster 7 in a pseudotime-dependent manner. (E) Map of the osteoclast activity score on UMAP. (F) Boxplot of the osteoclast activity scores of clusters 1 and 7.

Article Snippet: No antibody was added to the first tube, 5 μL of CD14 antibody (Proteintech, FITC-65056) was added to the second tube, 5 μL of CD16 antibody (Proteintech, PE65090) was added to the third tube, and 5 μL of CD14 antibody and 5 μL of CD16 antibody were added to the fourth tube.

Techniques: Plasmid Preparation, Expressing, Activity Assay

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Single-cell RNA sequencing reveals that an imbalance in monocyte subsets rather than changes in gene expression patterns is a feature of postmenopausal osteoporosis.

doi: 10.1093/jbmr/zjae065

Figure Lengend Snippet: Figure 3. Changes in proportions of cluster 1 and cluster 7 in unsorted bone marrow. (A) Cluster 1 monocytes were identified by AUCell in unsorted bone marrow cells. (B) Mapping the AUC score of cluster 1 on the tSNE scatter plot. (C) Cluster 7 monocytes were identified by AUCell in unsorted bone marrow cells. (D) Mapping the AUC score of cluster 7 on the tSNE scatter plot. (E) Clusters 1 and 7 identified by AUC were mapped on the tSNE scatter plot. (F) Unsupervised inference of developmental directions for monocytes using VECTOR. (G) Stacked column charts of the proportions of clusters 1 and 7 of bone marrow CD14+ monocytes in healthy postmenopausal women and women with PMOP. (H) Boxplot of the proportions of cluster 1 bone marrow CD14+ monocytes in healthy postmenopausal women and women with PMOP. (I) Boxplot of the proportions of cluster 7 bone marrow CD14+

Article Snippet: No antibody was added to the first tube, 5 μL of CD14 antibody (Proteintech, FITC-65056) was added to the second tube, 5 μL of CD16 antibody (Proteintech, PE65090) was added to the third tube, and 5 μL of CD14 antibody and 5 μL of CD16 antibody were added to the fourth tube.

Techniques: Plasmid Preparation

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Single-cell RNA sequencing reveals that an imbalance in monocyte subsets rather than changes in gene expression patterns is a feature of postmenopausal osteoporosis.

doi: 10.1093/jbmr/zjae065

Figure Lengend Snippet: Figure 4. Bulk transcriptome data did not detect differences, and genes in the same cluster were consistently expressed between the healthy and PMOP samples. (A) PCA of bulk transcriptome data of peripheral blood monocytes from 20 postmenopausal women with high BMD and 20 postmenopausal women with low BMD in the GSE56815 dataset. (B) Volcano plot of bulk transcriptome data of peripheral blood monocytes from 20 postmenopausal women with high BMD and 20 postmenopausal women with low BMD in the GSE56815 dataset. (C) PCA of bulk transcriptome data of peripheral blood monocytes from 5 postmenopausal women with high BMD and 5 postmenopausal women with low BMD in the GSE2208 dataset. (D) Volcano plot of bulk transcriptome data of peripheral blood monocytes from 5 postmenopausal women with high BMD and 5 postmenopausal women with low BMD in the GSE2208 dataset. (E) The scatter plot of gene expression of each gene processed by ln (X + 1) in the scRNA-seq data of monocytes sorted by CD14 microbeads. (F–L) Volcano plot of 7 clusters.

Article Snippet: No antibody was added to the first tube, 5 μL of CD14 antibody (Proteintech, FITC-65056) was added to the second tube, 5 μL of CD16 antibody (Proteintech, PE65090) was added to the third tube, and 5 μL of CD14 antibody and 5 μL of CD16 antibody were added to the fourth tube.

Techniques: Gene Expression

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Single-cell RNA sequencing reveals that an imbalance in monocyte subsets rather than changes in gene expression patterns is a feature of postmenopausal osteoporosis.

doi: 10.1093/jbmr/zjae065

Figure Lengend Snippet: Figure 5. Changes in proportions of peripheral blood CD14+ monocytes in cluster 1 and cluster 7. (A) The “BayesPrism” R package was used to determine the different cell clusters of each sample in the GSE56815 dataset. The z-score for cluster 1 of CD14+ monocytes was greater in postmenopausal women with high BMD than in postmenopausal women with low BMD. (B) The z-score for cluster 7 of CD14+ monocyte was lower in postmenopausal women with high BMD than in postmenopausal women with low BMD. (C) ROC curve of the z-scores for cluster 1 based on BayesPrism. (D) ROC curve of the z- scores for cluster 7 based on BayesPrism. (E) The z-score based on the ssGSEA) method for cluster 1 of CD14+ monocytes was greater in postmenopausal women with high BMD than in postmenopausal women with low BMD. (F) The z-score based on the ssGSEA method for cluster 7 of CD14+ monocyte was lower in postmenopausal women with high BMD than that in postmenopausal women with low BMD. (G) ROC curve of z-scores for cluster 1 based on ssGSEA. (H) ROC curve of z-scores for cluster 1 based on ssGSEA. (I) Expression of CD14 and CD16 in CD14+ cells from postmenopausal women with normal BMD determined by flow cytometry. (J) Expression of CD14 and CD16 in CD14+ cells from postmenopausal women with low BMD determined by flow cytometry. (K) The proportion of CD14+CD16+ cells in the peripheral blood of postmenopausal women with normal BMD or low BMD was determined by flow cytometry (n = 10 for normal BMD and n = 10 for low BMD). All flow cytometry results are provided as a “flow cytometry” file.

Article Snippet: No antibody was added to the first tube, 5 μL of CD14 antibody (Proteintech, FITC-65056) was added to the second tube, 5 μL of CD16 antibody (Proteintech, PE65090) was added to the third tube, and 5 μL of CD14 antibody and 5 μL of CD16 antibody were added to the fourth tube.

Techniques: Expressing, Cytometry

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Single-cell RNA sequencing reveals that an imbalance in monocyte subsets rather than changes in gene expression patterns is a feature of postmenopausal osteoporosis.

doi: 10.1093/jbmr/zjae065

Figure Lengend Snippet: Figure 6. Cluster 1 and cluster 7 of bone marrow CD14+ monocytes exhibited different cell–cell communications. (A) CellChat was used to quantify and visualize the global communication map between clusters 1 and 7 of bone marrow CD14+ monocytes and other cell groups. (B) Clusters 1 and 7 widely received ligands from other cell groups. (C) Clusters 1 and 7 sent ligands to other cell groups. (D) Interaction between ligands emitted by different cell groups and specific receptors in clusters 1 and 7. (E) Interaction between specific receptors in clusters 1 and 7 and ligands emitted by different cell groups. (F) Cluster 1 lacks ligand–receptor interactions from other distinct cell groups with MHC-II as a ligand, while cluster 7 has the opposite effect. (G) Significant differences in IL-16-CD4 interactions between cluster 1 and cluster 7. (H) Significant differences in RETN-CAP1 interactions between cluster 1 and cluster 7.

Article Snippet: No antibody was added to the first tube, 5 μL of CD14 antibody (Proteintech, FITC-65056) was added to the second tube, 5 μL of CD16 antibody (Proteintech, PE65090) was added to the third tube, and 5 μL of CD14 antibody and 5 μL of CD16 antibody were added to the fourth tube.

Techniques:

Journal: Journal of Cellular Physiology

Article Title: Alterations in CD39/CD73 axis of T cells associated with COVID‐19 severity

doi: 10.1002/jcp.30805

Figure Lengend Snippet: CD39 and CD73 expression in CD4 + (a) and CD8 + (b) T cells of a healthy noninfected donor after the incubation with plasma obtained from controls, mild coronavirus disease 2019 (COVID‐19) or severe COVID‐19 patients. In addition, peripheral blood mononuclear cell (PBMC) of severe COVID‐19 ( n = 3) were treated in vitro with adenosine and the activation of NF‐κBp65 were evaluated in CD3 + T cells (c) and CD14 + monocytes (d), as well as the production of interleukin (IL)‐1β (e), IL‐10 (f), IL‐17a (g), and tumor necrosis factor‐α (TNF‐α) (h) were evaluated. Data are presented as mean ± SD. Group comparisons were performed by one‐way analysis of variance with Bonferroni's post hoc test ( p ≤ 0.05).

Article Snippet: Then, cells were stained with 5 μl monoclonal antibodies (all antihuman) conjugated with specific fluorochromes: CD3 FITC (EbioScience) or CD14 FITC (BIOGEMS).

Techniques: Expressing, Incubation, Clinical Proteomics, In Vitro, Activation Assay

Journal: PLoS ONE

Article Title: Administration of BMSCs with Muscone in Rats with Gentamicin-Induced AKI Improves Their Therapeutic Efficacy

doi: 10.1371/journal.pone.0097123

Figure Lengend Snippet: A: Immunophenotype of isolated BMSCs. Isolated rat BMSCs were characterized by FACS. BMSCs were positive for CD29, CD44, CD73, CD90, CD105, and CD166, and nearly negative for CD14, CD34, and CD45. B: Differentiation characteristics of BMSCs. The phase contrast of BMSCs is shown on the left. Osteogenic differentiation was detected by AP staining (middle), and adipogenic differentiation was visualized by Oil Red O staining of the lipid vesicles (right). BMSCs cultured with normal medium were used as the negative control group for each stain (B1 and B2). C: Immunophenotype of isolated RTECs. The appearance of kidney tubules is shown on the left and the confluent RTECs in culture were shown in C1. The isolated rat RTECs were characterized by immunohistochemistry (middle) and FACS (right), and they were positive for CK-18. All scale bars correspond to 200 µm.

Article Snippet: The antibodies, including CD14 conjugated to FITC (Biorbyt, UK), CD29 conjugated to FITC (BD, USA), CD44 conjugated to FITC (BD), CD45 conjugated to PE (BD), and CD90 conjugated to FITC (BioLegend, USA), were added and incubated for 30 min at 4°C.

Techniques: Isolation, Staining, Cell Culture, Negative Control, Immunohistochemistry