Journal: Frontiers in Immunology

Article Title: The significance of CD16+ monocytes in the occurrence and development of chronic thromboembolic pulmonary hypertension: insights from single-cell RNA sequencing

doi: 10.3389/fimmu.2024.1446710

Figure Lengend Snippet: Changes in peripheral blood monocyte subsets in CTEPH patients. (A) UMAP plot of Mononuclear Phagocyte System (MPS) was divided into three cell types: monocytes, macrophages, and conventional dendritic cells (cDCs). Subsequently, monocytes were further divided into CD14+ monocytes and CD16+ monocytes. (B) A dot plot was used to annotate marker genes for CD14+ monocytes and CD16+ monocytes, along with a heatmap displaying the top 10 differentially expressed genes between the two cell types. (C) Ro/e (ratio of observed cell number to expected cell number) revealed the proportion of CD16+ monocytes in the CTEPH-N, CTEPH-I and HC groups. (D) The heat map showed the degree of correlation, and the numbers in the graph represented the correlation coefficient. (6MWD: 6-minute walk distance; PVR: pulmonary vascular resistance) (E) Schematic diagram of the gating strategy for distinguishing CD14+ monocytes from CD16+ monocytes by flow cytometry. (F) Differences in the proportion of CD16+ monocyte subsets in total monocytes between CTEPD patients ( n = 15) and matched healthy controls ( n = 15) were assessed by flow cytometry. All data were presented as means ± SEM, *** P < 0.001.

Article Snippet: Then the cells were resuspend in the flow cytometer wash buffer (2% FBS in PBS) and stained with the following antibodies according to the standard protocol: CD14-PE (Elabscience, E-AB-F1209D) and CD16-FITC (Elabscience, E-AB-F1236C) for monocyte labeling.

Techniques: Marker, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: The significance of CD16+ monocytes in the occurrence and development of chronic thromboembolic pulmonary hypertension: insights from single-cell RNA sequencing

doi: 10.3389/fimmu.2024.1446710

Figure Lengend Snippet: The functional characteristics of peripheral blood CD16+ monocytes in CTEPH patients. (A) GO analysis (biological process) of upregulated genes in CD16+ monocytes compared with CD14+ monocytes in CTEPH patients. (B) KEGG analysis of upregulated genes in CD16+ monocytes compared with CD14+ monocytes in CTEPH patients. (C) GSEA bar plot of CD16+ monocytes versus CD14+ monocytes in patients with CTEPH. (D) GO analysis (biological process) of upregulated genes in CD16+ monocytes between the CTEPH and healthy control samples. (E) KEGG analysis of upregulated genes in CD16+ monocytes between CTEPH patients and healthy controls. (F) GSVA heatmap of CD16+ monocytes between CTEPH patients and healthy controls. (G) Heat map of transcription factors upregulated in CD16+ monocytes between CTEPH patients and healthy controls.

Article Snippet: Then the cells were resuspend in the flow cytometer wash buffer (2% FBS in PBS) and stained with the following antibodies according to the standard protocol: CD14-PE (Elabscience, E-AB-F1209D) and CD16-FITC (Elabscience, E-AB-F1236C) for monocyte labeling.

Techniques: Functional Assay, Control

Journal: Frontiers in Immunology

Article Title: The significance of CD16+ monocytes in the occurrence and development of chronic thromboembolic pulmonary hypertension: insights from single-cell RNA sequencing

doi: 10.3389/fimmu.2024.1446710

Figure Lengend Snippet: Characteristics analysis of CD16+ monocytes differentiation into IL-1β+ macrophages in peripheral blood of CTEPH patients. (A) Heatmap shows the correlation of CD16+ monocytes with four macrophage subgroups. (B) Resident macrophage scores of four macrophage subgroups. (C) Immunofluorescence images of CD68 (red), IL-1β (green) and DAPI (blue) in inferior vena cava thrombus tissue at different time points (1 day, 7 days and 14 days after ligation). Scale bar = 300 µm. (D) Pseudotime analysis of the developmental trajectory of CD16+ monocytes and Macrophages 2. (E) The developmental trajectory of CD16+ monocytes and Macrophages 2 was clustered into three clusters. (F) Genes with significant expression changes along the pseudotime trajectory. (G) KEGG pathway enrichment analysis along the pseudotime of CD16+ monocytes differentiation into Macrophages 2.

Article Snippet: Then the cells were resuspend in the flow cytometer wash buffer (2% FBS in PBS) and stained with the following antibodies according to the standard protocol: CD14-PE (Elabscience, E-AB-F1209D) and CD16-FITC (Elabscience, E-AB-F1236C) for monocyte labeling.

Techniques: Immunofluorescence, Ligation, Expressing

Journal: PLoS ONE

Article Title: The Human Neutrophil Subsets Defined by the Presence or Absence of OLFM4 Both Transmigrate into Tissue In Vivo and Give Rise to Distinct NETs In Vitro

doi: 10.1371/journal.pone.0069575

Figure Lengend Snippet: A ) Neutrophils were immunofluorescently stained for OLFM4 (green) together with specific granule marker NGAL or azurophil granule marker CD63 (red), and DNA was labeled with DAPI (blue). Colocalization was analyzed by imaging flow cytometry and images show representative cells from the double positive populations (BF = brightfield). The diagram shows the mean colocalization score for the two fluorophores +SD from at least three experiments. The technical positive control was FITC-conjugated mouse anti-human CD16 antibody followed by Alexa Fluor 647-coupled goat anti-mouse secondary antibody and the technical negative control was FITC-conjugated mouse anti-human CD16 antibody together with DAPI, showing the minimum and maximum values that can be obtained by this analysis. The biological positive control was specific granule marker lactoferrin (LF) together with NGAL, and the biological negative control was lactoferrin together with CD63, showing the resolution of the analysis. B ) Pooled neutrophils from three donors were subjected to subcellular fractionation, and the fractions containing peak content of azurophil granule marker (MPO; fraction 1), specific granule marker (lactoferrin; fractions 10-12), gelatinase granule marker (gelatinase; fractions 13-15) and secretory vesicle marker (latent alkaline phosphatase; fraction 22) were subjected to Western blot with anti-OLFM4 antibody using rOLFM4 as a positive control. One representative blot out of three is shown. Lactoferrin (specific granule marker) and gelatinase (gelatinase granule marker) blots are also shown for fractions 10-15.

Article Snippet: CD177 was stained using a mouse anti-human CD177 antibody conjugated with phycoerythrin (Abcam), diluted 1:5, and CD16 with a mouse monoclonal anti-human CD16 antibody conjugated with fluorescein isothiocyanate (FITC) (EuroBioSciences, Friesoythe, Germany), diluted 1:20.

Techniques: Staining, Marker, Labeling, Imaging, Flow Cytometry, Positive Control, Negative Control, Fractionation, Western Blot

Journal: PLoS ONE

Article Title: The Human Neutrophil Subsets Defined by the Presence or Absence of OLFM4 Both Transmigrate into Tissue In Vivo and Give Rise to Distinct NETs In Vitro

doi: 10.1371/journal.pone.0069575

Figure Lengend Snippet: A ) Neutrophils isolated from heparinized blood were adhered to glass coverslips and either stimulated with PMA to form NETs (+PMA) or left unstimulated (-PMA). They were then left unpermeabilized (-Perm.) or permeabilized using acetone/methanol (+Perm.), after which they were immunostained for OLFM4 (red), plasma membranes (PM) were stained using FITC-conjugated WGA (green), and DNA was stained with DAPI (blue). Confocal images show representative cells or NETs from at least three individual experiments. B ) Adherent neutrophils were induced to form NETs as in A, and unpermeabilized samples were immunostained for OLFM4 (green) and MPO (red). DNA was stained with DAPI (blue). Confocal images show representative NETs from three individual experiments. A – B : Arrows indicate OLFM4-containing cells or NETs, while arrow heads indicate cells or NETs without OLFM4. The fluorophore conjugates used for each staining are indicated (AF = Alexa Fluor). The scale bars represent 5 µm.

Article Snippet: CD177 was stained using a mouse anti-human CD177 antibody conjugated with phycoerythrin (Abcam), diluted 1:5, and CD16 with a mouse monoclonal anti-human CD16 antibody conjugated with fluorescein isothiocyanate (FITC) (EuroBioSciences, Friesoythe, Germany), diluted 1:20.

Techniques: Isolation, Clinical Proteomics, Staining

Journal: American Journal of Translational Research

Article Title: The antiviral immune defense may be adversely influenced by weight loss through a calorie restriction program in obese women

doi:

Figure Lengend Snippet: Baseline characteristics of the intervention and control groups

Article Snippet: Antibodies for CD3/CD4/CD8 (FITC/R-PE/CyQ conjugates), CD3/CD19/CD45 (FITC/R-PE/CyQ conjugates), and CD3/CD16/56 (FITC/R-PE conjugates) were utilized (IQ Products, Netherlands).

Techniques:

Journal: American Journal of Translational Research

Article Title: The antiviral immune defense may be adversely influenced by weight loss through a calorie restriction program in obese women

doi:

Figure Lengend Snippet: Lymphocyte subset counts before and after the study in the control and intervention groups

Article Snippet: Antibodies for CD3/CD4/CD8 (FITC/R-PE/CyQ conjugates), CD3/CD19/CD45 (FITC/R-PE/CyQ conjugates), and CD3/CD16/56 (FITC/R-PE conjugates) were utilized (IQ Products, Netherlands).

Techniques: