Journal: medRxiv

Article Title: Longitudinal multi-omics analysis identifies responses of megakaryocytes, erythroid cells and plasmablasts as hallmarks of severe COVID-19 trajectories

doi: 10.1101/2020.09.11.20187369

Figure Lengend Snippet: a , Schematic workflow of the scRNA-seq analyses performed on the B cell compartment identified in . b , B cell compartment subtypes represented as a UMAP. In total, 22,190 cells are depicted. Clusters represented are Memory B cells (MB, dark red), naïve B cells (N, red), transitional B cells (trans-orange) and plasmablast (PB, blue). c , B cell compartment pseudotimes represented as a UMAP. d , Flow cytometry analysis of B cell subtypes. CD19 + B cells were stained for CD20 and CD27. CD20 - CD27 high B cells classified as PB, CD20 + CD27 + cells as MB and CD20 + CD27 - cells as N. Proportions of each cell type among CD19 + B cells were plotted against the sampling time relative to the disease onset. The points were coloured by corresponding pseudotime and connected by patient. Only patients from Kiel cohort ( n = 7 individuals) are shown. e , Plasmablast-specific UMAP highlighted neutrophil-like cells (NL). Smaller UMAPs corresponding to expression of plasmablast markers ( CD27 , CD38 , and TNFRSF17 ) and neutrophil-like markers ( ELANE , MPO , and CAMP ). f , Cell trajectory analysis of B cell compartment. Cell trajectory was calculated using Monocle3 and B cell subtypes are displayed in left panel followed by the corresponding transition-pseudotime analysis rooted on transitional B cells (purple) and differentiated into 2 branches: B cells naïve/memory branch (grey line/culminating in yellow) and an over imposed plasmablast branch, from which most of the plasmablasts (black line/culminating in orange) are not connected to neutrophil-like cells (grey). g , Dot plot for pseudotime signature genes in plasmablasts. Genes were selected based on ten most characteristic upregulated genes. Colour discriminates upregulated (red) or downregulated (blue) genes, while point sizes represents the number of cells per group expressing the corresponding gene. h , GO enrichment analysis for upregulated genes during disease trajectory. Size of the dots is proportional to the gene ratio and the colour corresponds to the p -value of the enrichment. Selected top terms are visualized. i , Gene expression of genes of interest in B cell subtypes, pseudotimes and cohort 2. Genes of interest selected based on their high expression in PB or NL and upregulated during disease ( DERL3 , CD38, PIM2, IFI6 , XBP1 , and SLC1A4 ). For each gene, top violin plot depicting B cell subtypes expression, centre violin plot based on pseudotime expressions and bottom violin plot based on the expression of cohort 2 divided by disease classification, healthy control (white), mild disease (light grey) and severe disease (dark grey). j , Metabolic pathways enriched in B cell compartment subtypes. Top 20 active metabolic pathways for context-specific metabolic networks reconstructed in plasmablasts are shown. For each B cell subtype, significant differences in metabolic activity were determined using a Kruskal-Wallis test. Number of reaction counts found per pathways is displayed as colour intensity.

Article Snippet: B cell subsets were stained using antibodies against CD19 (clone REA675, Miltenyi), CD20 (clone REA780, Miltenyi), CD27 (clone M-T271, Biolegend, San Diego, California, US), CD138 (clone BB4/MI15, Biolegend), HLA-DR (clone REA332, Miltenyi), IgD (clone REA740, Miltenyi), IgM (clone MHM-88, Biolegend), IgA (clone M24A, Merck Millipore), CD95 (clone DX2, Biolegend) as well as CD3 (clone OKT3, Biolegend) and CD14 (clone M5E2, Biolegend) for the dump channel.

Techniques: Flow Cytometry, Staining, Sampling, Expressing, Gene Expression, Control, Activity Assay

Journal: Mediators of Inflammation

Article Title: Relationship between Helicobacter pylori Infection and Plasmacytoid and Myeloid Dendritic Cells in Peripheral Blood and Gastric Mucosa of Children

doi: 10.1155/2019/7190596

Figure Lengend Snippet: Gating strategy for multicolor flow cytometric detection of total dendritic cells and their subsets. At first, leukocytes were gated by forward scatter characteristic (FSC) versus side scatter characteristics (SSC). Next, doublets and aggregates were avoided by selecting singlet cells on a forward scatter: FSC (area)/FSC (height) plot. Then total dendritic cells (tDCs) were gated for fluorescence intensity of HLA-DR peridinin-chlorophyll proteins (PerCP) versus lineage-1 fluorescein (FITC), and using the fluorescence intensity of CD11c phycoerytrin (PE) or CD123 PE, myeloid dendritic cells (mDCs) or plasmacytoid dendritic cells (pDCs) were gated (open histrograms). Then in tDCs and their subsets, a percentage of CD83 + and CD86 + cells was gated (open histograms.) The grey line represents the staining profile for fluorescence minus one (FMO) control.

Article Snippet: First, two tubes containing 200 μ L whole blood were stained with FITC Lin-1 lineage cocktail (antibodies against human CD3, CD14, CD16, CD19, CD20, and CD56), PerCP-anti-human HLA-DR, APC-anti-human CD83, PE-Cy7-anti-human CD86, PE-anti-human CD11c, or PE-anti-human CD123 (BD Biosciences, Franklin Laker, New Jersey, USA) antibodies for 30 min at room temperature (RT).

Techniques: Fluorescence, Staining

Journal: Antioxidants

Article Title: Effect of Heme Oxygenase-1 on Melanoma Development in Mice—Role of Tumor-Infiltrating Immune Cells

doi: 10.3390/antiox9121223

Figure Lengend Snippet: Effect of HO-1 expression in mice on leukocytes infiltration in primary tumors. ( A ) Percentage of leukocytes (CD45 + cells) in tumors. Cytometric analysis. Each point represents mean ± SE ( N = 3–9). * p < 0.05 vs. HO-1 +/+ mice, # p < 0.05 ♂ vs. ♀. ( B ) Subpopulations of infiltrating leukocytes: CD11b + Gr1 + myeloid cells, CD11b + Gr1 − myeloid cells, CD3 + T lymphocytes; CD19 + B lymphocytes; Nk 1.1 + NK cells. Cytometric analysis.

Article Snippet: Antibodies against CD3, CD11b, CD19, CD45, Gr-1, and NK 1.1, the BD™ Cytometric Bead Array (CBA) Mouse Inflammation kit and 2,2′,2″,2‴-(Ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA) were purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Expressing

Journal: Antioxidants

Article Title: Effect of Heme Oxygenase-1 on Melanoma Development in Mice—Role of Tumor-Infiltrating Immune Cells

doi: 10.3390/antiox9121223

Figure Lengend Snippet: Effect of HO-1 expression in mice on concentrations of proangiogenic and proinflammatory cytokines in sera after intracutaneous injection with melanoma cells. ( A ) Concentration of IFN-γ. Cytometric analysis. ( B ) Concentration of keratinocyte chemoattractant (KC). ELISA. ( C ) Concentration of IL-10. Cytometric analysis. ( D ) Concentration of IL-6. Cytometric analysis. ( E ) Concentration of vascular endothelial growth factor (VEGF). Cytometric analysis. ( F ) Concentration of IL-12. Cytometric analysis. ( G ) Concentration of MCP-1. ELISA. Each bar represents mean ± SE ( N = 4–10; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. HO-1 +/+ mice, # p < 0.05, ## p < 0.01♂ vs. ♀).

Article Snippet: Antibodies against CD3, CD11b, CD19, CD45, Gr-1, and NK 1.1, the BD™ Cytometric Bead Array (CBA) Mouse Inflammation kit and 2,2′,2″,2‴-(Ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA) were purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Expressing, Injection, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Antioxidants

Article Title: Effect of Heme Oxygenase-1 on Melanoma Development in Mice—Role of Tumor-Infiltrating Immune Cells

doi: 10.3390/antiox9121223

Figure Lengend Snippet: Effect of HO-1 expression in mice on concentrations of proangiogenic and proinflammatory cytokines in sera after intravenous injection with melanoma cells. ( A ) Concentration of IFN-γ. Cytometric analysis. ( B ) Concentration of KC. ELISA. ( C ) Concentration of VEGF. ELISA. ( D ) Concentration of IL-10. Cytometric analysis. ( E ) Concentration of IL-12. Cytometric analysis. ( F ) Concentration of MCP-1. Cytometric analysis. ( G ) Concentration of IL-6. Cytometric analysis. Each bar represents mean ± SE ( N = 3–8; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. HO-1 +/+ mice, # p < 0.05, ♂ vs. ♀).

Article Snippet: Antibodies against CD3, CD11b, CD19, CD45, Gr-1, and NK 1.1, the BD™ Cytometric Bead Array (CBA) Mouse Inflammation kit and 2,2′,2″,2‴-(Ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA) were purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Expressing, Injection, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Stem cells international

Article Title: Feasibility and Efficiency of Human Bone Marrow Stromal Cell Culture with Allogeneic Platelet Lysate-Supplementation for Cell Therapy against Stroke.

doi: 10.1155/2016/6104780

Figure Lengend Snippet: Figure 4: Flow cytometric analysis of the surface markers. Gray lines: each specific antibody (CD44, CD90, CD105, CD106, CD146, CD166, CD19, and CD45); black lines: each isotopic antibody.

Article Snippet: The hBMSCs cultured with PL in CPC were suspended with PBS containing 3% FCS.They were incubated with either a mouse monoclonal antibody against human CD19 (R&D Systems; dilution, 1 : 100), CD44 (R&D Systems; 1 : 100), CD45 (R&D Systems; 1 : 100), CD90 (R&D Systems; 1 : 100), CD105 (R&D Systems; 1 : 100), CD106 (R&D Systems; 1 : 100), CD146 (R&D Systems; 1 : 100), CD166 (R&D Systems; 1 : 100), or each mouse isotypic control for 30min on ice.

Techniques: