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: FIGURE 2 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-ABF1209D) and CD16-FITC (Elabscience, E-AB-F1236C) for monocyte labeling.

Techniques: Marker, 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: FIGURE 3 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-ABF1209D) and CD16-FITC (Elabscience, E-AB-F1236C) for monocyte labeling.

Techniques: Functional Assay, Control

Journal: Cancer Reports

Article Title: The effects of CD148 Q276P / R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling

doi: 10.1002/cnr2.1566

Figure Lengend Snippet: Generation of A431D cells that stably express CD148 forms (WT, Q276P/R326Q). (A) DNA sequence of mutated CD148 cDNA, confirming Q276P and R326Q mutations. (B) CD148 surface expression were examined in A431D‐CD148 (WT, Q276P/R326Q) cells and the control A431D cells that was generated by the infection of empty retrovirus (Mock) by flow cytometry using a PE‐conjugated anti‐CD148 antibody. Representative results of four independent experiments are shown. Mean fluorescence intensity is also shown. The fluorescence intensity of CD148 WT cells is expressed as 1.0. A431D‐CD148 WT and Q276P/R326Q cells express comparable levels of CD148. A431D‐Mock cells show no CD148 expression. (C) Expression of CD148 in A431D‐CD148 (WT, Q276P/R326Q) cells was also examined by Western blotting using anti‐CD148 or anti‐HA antibodies. Fifty micrograms of protein cell lysates were loaded into each well. The loading of proteins was assessed by anti‐β actin Western blot. The ratios of CD148 or HA to β actin are also shown. The ratios in A431D‐CD148 WT cells are expressed as 1.0. Representative results of four independent experiments are shown. (D) Immunolocalization of CD148 or HA epitope (both red) were examined in A431D‐Mock and A431D‐CD148 (WT, Q276P/R326Q) cells. The antibody against CD148 extracellular domain was used. Representative results of four independent experiments are shown. Scale bar = 25 μm. CD148 WT and Q276P/R326Q are similarly localized to the cell membrane (arrowheads). In anti‐HA immunostaining, there is perinuclear CD148 presence that is generally observed in stable cells

Article Snippet: The cells were dissociated with 0.05%Trypsin, 0.53 mM EDTA (Corning, Manassas, VA), washed with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich), resuspended in cold 0.5% BSA‐PBS, and 2 × 10 5 cells were incubated with 5 μl of PE‐conjugated anti‐human CD148 mouse monoclonal (clone 143‐41, R&D Systems) or 10 μl of PE‐conjugated anti‐human EGFR (BD Biosciences) for 45 min at 4°C.

Techniques: Stable Transfection, Sequencing, Expressing, Control, Generated, Infection, Flow Cytometry, Fluorescence, Western Blot, Membrane, Immunostaining

Journal: Cancer Reports

Article Title: The effects of CD148 Q276P / R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling

doi: 10.1002/cnr2.1566

Figure Lengend Snippet: Cell proliferation rate in A431D‐CD148 (WT, Q276P/R326Q) cells. Cells (1.0 × 10 3 ) were plated in 96‐well plates and serum starved (0.1% FBS) for overnight (day 1). Cells were then cultured in growth medium supplemented with 2.5% FBS. Cell number was assessed at day 1, 2, 3 and 4. Data are means ± SEM of quadruplicate determinations. Representative data of four independent experiments is shown. ** p < .01. A431D‐CD148 (WT, Q276P/R326Q) cells showed lower cell proliferation rates than A431D‐Mock cells. No significant difference was observed between A431D‐CD148 WT and Q276P/R326Q cells

Article Snippet: The cells were dissociated with 0.05%Trypsin, 0.53 mM EDTA (Corning, Manassas, VA), washed with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich), resuspended in cold 0.5% BSA‐PBS, and 2 × 10 5 cells were incubated with 5 μl of PE‐conjugated anti‐human CD148 mouse monoclonal (clone 143‐41, R&D Systems) or 10 μl of PE‐conjugated anti‐human EGFR (BD Biosciences) for 45 min at 4°C.

Techniques: Cell Culture

Journal: Cancer Reports

Article Title: The effects of CD148 Q276P / R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling

doi: 10.1002/cnr2.1566

Figure Lengend Snippet: Expression of epidermal growth factor receptor (EGFR) and EGF‐induced ERK phosphorylation in A431D‐CD148 (WT, Q276P/R326Q) cells. (A) Cell surface expression of EGFR in A431D‐CD148 (WT and Q276P/R326Q) and A431D‐Mock (control) cells were examined by flow cytometry using a PE‐conjugated anti‐human EGFR antibody. HEK293 cells that do not express EGFR were used as a negative control. Mean fluorescence intensity is also shown (bottom right). The fluorescence intensity of A431D‐Mock cells is expressed as 1.0. Representative results of three independent experiments are shown. All three A431D cells showed comparable level of EGFR expression. (B) EGF‐induced ERK1/2 phosphorylation was examined in A431D‐CD148 (WT and Q276P/R326Q) and A431D‐Mock cells by ELISA as described in Section . Cells were treated with 10 ng/ml EGF for 5, 10, 15, and 30 min. The cells cultured in serum starved (SS) condition (0.1% FBS medium) for 30 min were used as a control. The p‐ERK/ERK ratios were normalized to serum‐starved (30 min) A431D‐Mock cells. Representative results of five independent experiments are shown. Data are means ± SEM of quadruplicate measurements. *** p < .001

Article Snippet: The cells were dissociated with 0.05%Trypsin, 0.53 mM EDTA (Corning, Manassas, VA), washed with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich), resuspended in cold 0.5% BSA‐PBS, and 2 × 10 5 cells were incubated with 5 μl of PE‐conjugated anti‐human CD148 mouse monoclonal (clone 143‐41, R&D Systems) or 10 μl of PE‐conjugated anti‐human EGFR (BD Biosciences) for 45 min at 4°C.

Techniques: Expressing, Phospho-proteomics, Control, Flow Cytometry, Negative Control, Fluorescence, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cancer Reports

Article Title: The effects of CD148 Q276P / R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling

doi: 10.1002/cnr2.1566

Figure Lengend Snippet: Immunoblotting to assess EGF‐induced epidermal growth factor receptor (EGFR) and ERK1/2 phosphorylation in A431D‐CD148 (WT, Q276P/R326Q) cells. (A) and (B) EGF‐induced EGFR and ERK1/2 phosphorylation was examined in A431D‐CD148 (WT, Q276P/R326Q) and A431D‐Mock cells by Western blotting as described in Section . Cells were plated in 100 mm dishes, serum was reduced (0.5% FBS), then cells were treated with 10 ng/ml EGF for 5, 10, 15, 30 min. The cells cultured in serum starved (SS) condition (0.1% FBS medium) for 30 min were used as a negative control. The p‐EGFR/EGFR ratios were normalized to A431D‐Mock cells treated with EGF for 5 min as EGFR phosphorylation was undetectable in serum starved (0.1% FBS) cells. The p‐ERK/ERK ratios were normalized to serum‐starved A431D‐Mock cells. Representative results of five independent experiments are shown

Article Snippet: The cells were dissociated with 0.05%Trypsin, 0.53 mM EDTA (Corning, Manassas, VA), washed with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich), resuspended in cold 0.5% BSA‐PBS, and 2 × 10 5 cells were incubated with 5 μl of PE‐conjugated anti‐human CD148 mouse monoclonal (clone 143‐41, R&D Systems) or 10 μl of PE‐conjugated anti‐human EGFR (BD Biosciences) for 45 min at 4°C.

Techniques: Western Blot, Phospho-proteomics, Cell Culture, Negative Control

Journal: Cancer Reports

Article Title: The effects of CD148 Q276P / R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling

doi: 10.1002/cnr2.1566

Figure Lengend Snippet: EGF‐induced cell proliferation in A431D‐CD148 (WT, Q276P/R326Q) cells. Cells (1.2 × 10 3 ) were plated in 96‐well plates (day 0), then serum starved (0.1% FBS) for overnight (day 1). Cells were then treated with 60 ng/ml, 120 ng/ml, and 240 ng/ml EGF in growth medium supplemented with 0.3% FBS. Cell number was assessed at day 1 (before EGF stimulation) and day 3. Representative results of four independent experiments are shown. Data are means ± SEM of quadruplicate determinations. ** p < .01, * p < .05

Article Snippet: The cells were dissociated with 0.05%Trypsin, 0.53 mM EDTA (Corning, Manassas, VA), washed with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich), resuspended in cold 0.5% BSA‐PBS, and 2 × 10 5 cells were incubated with 5 μl of PE‐conjugated anti‐human CD148 mouse monoclonal (clone 143‐41, R&D Systems) or 10 μl of PE‐conjugated anti‐human EGFR (BD Biosciences) for 45 min at 4°C.

Techniques:

Journal: Cancer Reports

Article Title: The effects of CD148 Q276P / R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling

doi: 10.1002/cnr2.1566

Figure Lengend Snippet: Effects of TSP1 on cell proliferation in A431D‐CD148 (WT, Q276P/R326Q) cells. Cells (1.0 × 10 3 ) were plated in 96‐well plates (day 0), then serum starved (0.1% FBS) for overnight (day 1). Cells were then treated with 1, 10, 20 nM of TSP1 in growth medium supplemented with 2.5% FBS. Cell number was assessed at day 1 (before TSP1 stimulation) and day 4. Representative results of four independent experiments are shown. Data are means ± SEM of quadruplicate determinations. ** p < .01

Article Snippet: The cells were dissociated with 0.05%Trypsin, 0.53 mM EDTA (Corning, Manassas, VA), washed with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich), resuspended in cold 0.5% BSA‐PBS, and 2 × 10 5 cells were incubated with 5 μl of PE‐conjugated anti‐human CD148 mouse monoclonal (clone 143‐41, R&D Systems) or 10 μl of PE‐conjugated anti‐human EGFR (BD Biosciences) for 45 min at 4°C.

Techniques:

Journal: Pediatric Rheumatology Online Journal

Article Title: Surface expression and genotypes of Toll- like receptors 2 and 4 in patients with juvenile idiopathic arthritis and systemic lupus erythematosus

doi: 10.1186/1546-0096-11-9

Figure Lengend Snippet: Representative flow cytometry histograms showing (a) TLR2 and (b) TLR4 expression on CD14 + monocytes. Blue filled histograms: isotype controls and green or red filled histogram TLR expression. Mean channel fluorescence intensity (MFI) derived from fluorescence histogram was used to study the level of cell surface TLR expression. Delta MFI (dMFI) was calculated as a subtraction and recorded as the MFI of the TLR2 or TLR4 antibody minus the MFI of the isotype-matched control antibody.

Article Snippet: For surface staining 100 μl of whole blood samples were incubated with the following anti-human primary antibodies for 15 minutes in the dark at 4°C: anti-CD14 monoclonal antibody (mAb; 10 μl per 100 μl blood, FITC or PE labelled, clone 8 G3; Diaclone, Besançon, France), anti-TLR2 mAb (10 μl, FITC labelled, clone HTA 125; eBioscience, San Diego, CA) or TLR4 mAb (10 μl, PE labelled, clone HTA 125; eBioscience, San Diego, CA).

Techniques: Flow Cytometry, Expressing, Fluorescence, Derivative Assay, Control

Journal: Pediatric Rheumatology Online Journal

Article Title: Surface expression and genotypes of Toll- like receptors 2 and 4 in patients with juvenile idiopathic arthritis and systemic lupus erythematosus

doi: 10.1186/1546-0096-11-9

Figure Lengend Snippet: (a) Mean fluorescence intensity (dMFI) of TLR2-expression on CD14 + monocytes of patients with JIA, SLE and healthy controls. (b) Mean fluorescence intensity (dMFI) of TLR4-expression on CD14 + monocytes of patients with JIA, SLE and healthy controls.

Article Snippet: For surface staining 100 μl of whole blood samples were incubated with the following anti-human primary antibodies for 15 minutes in the dark at 4°C: anti-CD14 monoclonal antibody (mAb; 10 μl per 100 μl blood, FITC or PE labelled, clone 8 G3; Diaclone, Besançon, France), anti-TLR2 mAb (10 μl, FITC labelled, clone HTA 125; eBioscience, San Diego, CA) or TLR4 mAb (10 μl, PE labelled, clone HTA 125; eBioscience, San Diego, CA).

Techniques: Fluorescence, Expressing

Journal: Pediatric Rheumatology Online Journal

Article Title: Surface expression and genotypes of Toll- like receptors 2 and 4 in patients with juvenile idiopathic arthritis and systemic lupus erythematosus

doi: 10.1186/1546-0096-11-9

Figure Lengend Snippet: (a) Mean fluorescence intensity (dMFI) of TLR2-expression on CD14 + monocytes of patients in various disease phases compared to TLR2-expression on monocytes of healthy controls. (b) Mean fluorescence intensity (dMFI) of TLR4-expression on CD14 + monocytes of patients in various disease phases compared to TLR4-expression on monocytes of healthy controls.

Article Snippet: For surface staining 100 μl of whole blood samples were incubated with the following anti-human primary antibodies for 15 minutes in the dark at 4°C: anti-CD14 monoclonal antibody (mAb; 10 μl per 100 μl blood, FITC or PE labelled, clone 8 G3; Diaclone, Besançon, France), anti-TLR2 mAb (10 μl, FITC labelled, clone HTA 125; eBioscience, San Diego, CA) or TLR4 mAb (10 μl, PE labelled, clone HTA 125; eBioscience, San Diego, CA).

Techniques: Fluorescence, Expressing

Journal: Journal of Extracellular Biology

Article Title: Monocyte derived large extracellular vesicles in polytrauma

doi: 10.1002/jex2.70005

Figure Lengend Snippet: List of antibodies and isotypes used for cell‐ and cell derived large EV flow cytometric analysis. The antibodies were titrated against their matching isotype controls prior use and applied according to the manufacturers’ instructions.

Article Snippet: CD14 (mouse IgG2a) , VioBlue , 130‐094‐364 , Miltenyi Biotec , 0.01 , 1:50.

Techniques: Derivative Assay

Journal: Journal of Extracellular Biology

Article Title: Monocyte derived large extracellular vesicles in polytrauma

doi: 10.1002/jex2.70005

Figure Lengend Snippet: Antibody validation on cultured THP‐1 cells for EV detection and general used large EV gating strategy . (a) In vitro human THP‐1 monocyte cells were treated with PMA alone or PMA & LPS (each n ≥ 3), increasing CD14 expression. (b) Expression of CD14 on surface of AnnV + large EVs (lEVs) derived from non‐stimulated THP‐1 cells and from PMA alone or PMA & LPS stimulated THP‐1 cells. AnnV is used as a general large EV marker. Values given as mean with SEM. Three column statistical analysis was done by Kruskal–Wallis test including Dunn's post hoc test for multiple comparisons (*, p < 0.05; **, p < 0.01; ***, p < 0.001). (c) Thresholds testing for cell culture and serum derived large EV detection and gating. Prior use of flow cytometry gates sensitivity of used flow cytometer was set taking advantage of 1000, 500 and not detectable 200 nm beads. (d) Gating strategy for the detection of CD14‐APC on AnnV + large EVs controlled by matching REA isotype control.

Article Snippet: CD14 (mouse IgG2a) , VioBlue , 130‐094‐364 , Miltenyi Biotec , 0.01 , 1:50.

Techniques: Biomarker Discovery, Cell Culture, In Vitro, Expressing, Derivative Assay, Marker, Flow Cytometry, Control

Journal: Journal of Extracellular Biology

Article Title: Monocyte derived large extracellular vesicles in polytrauma

doi: 10.1002/jex2.70005

Figure Lengend Snippet: Semi quantification of human serum derived AnnV + CD14 + large EVs . (a) Workflow SOP for isolation of large EVs (lEVs) from 1 mL of human serum collected from polytrauma patients (ISS > 15, at day 0, at day 1 and day 7 post trauma). (b) AnnV + CD14 + large EVs counts in small EVs in polytrauma (ISS > 15) with internal organ damage (polytrauma OD) versus polytrauma (ISS ≥ 15) without internal organ damage (polytrauma w/o OD) upon admission (day 0). (c) AnnV + CD14 + large EVs counts in polytrauma (ISS > 15) polytrauma OD versus polytrauma w/o OD (ISS > 15) 24 h post trauma (day 1). (d) Day 1, corresponding AUROC, sensitivity, specificity, and cut‐off value, respectively. (e) AnnV + CD14 + large EVs counts in small EVs in polytrauma (ISS ≥ 15) polytrauma OD versus polytrauma w/o OD (ISS ≥ 15) 7 days post trauma (day 7). Values given as median with 95% CI. Three column statistical analysis was done by Kruskal–Wallis test including Dunn's post hoc test for multiple comparisons (*, p < 0.05; **, p < 0.01; ***, p < 0.001; not significant, n as indicated). Horizontal dotted line in panels B, C and E indicates calculated associated cut‐off (summarized in Table ).

Article Snippet: CD14 (mouse IgG2a) , VioBlue , 130‐094‐364 , Miltenyi Biotec , 0.01 , 1:50.

Techniques: Derivative Assay, Isolation

Journal: Journal of Extracellular Biology

Article Title: Monocyte derived large extracellular vesicles in polytrauma

doi: 10.1002/jex2.70005

Figure Lengend Snippet: AnnV + CD14 + large EVs kinetics at day 1 and day 7 in polytrauma (ISS > 15) and correlation with the Injury Severity Score (ISS) . (a) depicted are pairs of AnnV + CD14 + large EVs (lEVs) measured at day 1 and day 7 of enrolled polytrauma (ISS > 15). Values given as median. Statistical significance was assessed by two tailed paired t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). (b) Spearman ( r sp ) correlation between percentages of AnnV + CD14 + large EVs and ISS ( n = 24). p and r sp values as indicated.

Article Snippet: CD14 (mouse IgG2a) , VioBlue , 130‐094‐364 , Miltenyi Biotec , 0.01 , 1:50.

Techniques: Two Tailed Test

Journal: Journal of Extracellular Biology

Article Title: Monocyte derived large extracellular vesicles in polytrauma

doi: 10.1002/jex2.70005

Figure Lengend Snippet: Summarized data FACS of AnnV + CD14 + large EVS in polytrauma including associated SEM, percentage change and statistics at day 1.

Article Snippet: CD14 (mouse IgG2a) , VioBlue , 130‐094‐364 , Miltenyi Biotec , 0.01 , 1:50.

Techniques:

Journal: Journal of Extracellular Biology

Article Title: Monocyte derived large extracellular vesicles in polytrauma

doi: 10.1002/jex2.70005

Figure Lengend Snippet: List of antibodies used for human derived large EV surface marker analysis. The antibody was added according to manufacturer's recommendations and titrated against the matching isotype control prior use.

Article Snippet: CD14 (mouse IgG2a) , VioBlue , 130‐094‐364 , Miltenyi Biotec , 0.01 , 1:50.

Techniques: Derivative Assay, Marker, Control