pe conjugated mouse anti human tie2 antibody  (R&D Systems)

Journal: Cardiovascular Diabetology

Article Title: Increased frequency of proangiogenic tunica intima endothelial kinase 2 (Tie2) expressing monocytes in individuals with type 2 diabetes mellitus

doi: 10.1186/s12933-022-01497-6

Figure Lengend Snippet: Tie2 + monocyte gating and quantification. Representative fluorescence-activated cell sorting plots and gating strategy for the quantification of Tie2 + monocytes in healthy control individuals ( a ) and individuals with type 2 diabetes mellitus (T2DM) ( b ). Left 4 panels: Gating of Tie2 + monocytes within each monocyte subset, based on isotype controls. Right panel: Graph of Tie2 + monocyte frequency within the respective monocyte subset. No differences in Tie2 + monocyte frequency within subsets were observed in healthy control individuals. Within individuals with T2DM, the percentage of Tie2 + cells was significantly higher within the intermediate monocyte subset compared to the other monocyte populations. The percentage of Tie2 + monocytes within the classical monocyte population was similar between healthy individuals and individuals with T2DM ( c ). The percentage of Tie2 + cells within in the intermediate monocyte population was 1.9-fold higher in individuals with T2DM compared to healthy control individuals ( d ). The percentage of Tie2 + monocytes within in the non-classical monocyte population in individuals with T2DM was increased compared to healthy control individuals, however, without reaching the level of statistical significance (p = 0.056) ( e )

Article Snippet: In a subset of samples, we performed flow cytometry for TEMs using PE-conjugated mouse-α-human TIE2 (Clone 83,715, R&D Systems, McKinley Place NE, MN, USA) in a cocktail with the abovementioned antibodies against CD14 and CD16.

Techniques: Fluorescence, FACS, Control

Journal: JOR Spine

Article Title: The effects of 3D culture on the expansion and maintenance of nucleus pulposus progenitor cell multipotency

doi: 10.1002/jsp2.1131

Figure Lengend Snippet: Overview of all genes used for RT‐Q‐PCR in this study

Article Snippet: Tie2 + cells were sorted using FACS as previously described., , Freshly isolated NPCs (N = 3) were incubated with an anti‐human Tie2 PE‐conjugated monoclonal mouse antibody (#FAB3131P, clone 83 715, R&D systems) for 30 minutes on ice in 100 μL of FACS buffer ([PBS] with 0.5% bovine serum albumin [BSA] and 1 mM EDTA, all from Sigma‐Aldrich), protected from light.

Techniques:

Journal: JOR Spine

Article Title: The effects of 3D culture on the expansion and maintenance of nucleus pulposus progenitor cell multipotency

doi: 10.1002/jsp2.1131

Figure Lengend Snippet: Analysis of NP cells after the first and second phase of expansion. A, Schematic representation of the first phase of expansion of nucleus pulposus (NP) cells. Briefly, after dissection and digestion of the NP tissue from the intervertebral disk (IVD), NP cells were seeded in two‐dimensional (2D) or three‐dimensional (3D) into alginate beads. B‐H, Quantification by flow cytometry analysis of the amount of NP cells positive for different markers after culture in 2D or 3D for 1 week of culture. B‐H, Percentage of NP cells positive in 2D or 3D for, B, CD90 marker, C, CD73 marker, D, CD105 marker, E, CD45 marker, F, CD34 marker, G, CD146 marker, and, H, Tie2 marker. I‐L, Quantification of the relative gene on NP cells for different genes after culture in 2D or 3D for 1 week of culture. I, Relative gene expression of NANOG in NP cells in 2D or 3D. J, Relative gene expression of SOX2 in NP cells in 2D or 3D. K, Relative gene expression of OCT4 in NP cells in 2D or 3D. L, Relative gene expression of TEK in NP cells in 2D or 3D. M, Schematic representation of the second phase of expansion of NP cells. Briefly, after the first phase of expansion, NP cells cultured in 2D or 3D were putting back in the fibronectin‐coated flask/surface. N‐S, Quantification by flow cytometry analysis of the amount of NP cells (previously cultivated in 2D or 3D) positive for different markers after culture in the fibronectin‐coated surface for 1 week of culture for, N, CD90 marker, O, CD73 marker, P, CD105 marker, Q, CD45 marker, R, CD34 marker, and, S, Tie2 marker. T‐W, Quantification of the relative gene on NP cells (previously cultivated in 2D or 3D) for different genes after culture on the fibronectin‐coated surface. T, Relative gene expression of NANOG in NP cells. U, Relative gene expression of SOX2 in NP cells. V, Relative gene expression of OCT4 in NP cells. W, Relative gene expression of TEK in NP cells (n = 6) for each gene. Data are presented as mean ± SD. NP cells cultured in 2D during the first expansion phase = blue bars. NP cells cultured in 3D during the first expansion phase = red bars. Tie2, angiopoietin‐1 receptor; NANOG , homeobox protein NANOG; SOX2 , SRY (sex‐determining region Y)‐box 2; OCT4 , octamer‐binding transcription factor 4; TEK , angiopoietin‐1 receptor

Article Snippet: Tie2 + cells were sorted using FACS as previously described., , Freshly isolated NPCs (N = 3) were incubated with an anti‐human Tie2 PE‐conjugated monoclonal mouse antibody (#FAB3131P, clone 83 715, R&D systems) for 30 minutes on ice in 100 μL of FACS buffer ([PBS] with 0.5% bovine serum albumin [BSA] and 1 mM EDTA, all from Sigma‐Aldrich), protected from light.

Techniques: Dissection, Flow Cytometry, Marker, Gene Expression, Cell Culture, Binding Assay

Journal: JOR Spine

Article Title: The effects of 3D culture on the expansion and maintenance of nucleus pulposus progenitor cell multipotency

doi: 10.1002/jsp2.1131

Figure Lengend Snippet: Comparison of nucleus pulposus (NP) cells Tie2 + and NP cells Tie2 − . A, Schematic representation of the process leading to the comparison of NP cells Tie2 + and NP cells Tie2 − . Briefly, after dissection and digestion of the NP tissue from intervertebral disk (IVD), NP cells were sorted for angiopoietin‐1 receptor (Tie2) marker. Positive (Tie2 + ) and negative (Tie2 − ) cells were seeded in two‐dimensional (2D) or in three‐dimensional (3D) (into alginate beads) and analyzed. A,B, Quantification of cell metabolism by Alamar Blue assay on Tie2 + and Tie2 − NP cells (cultivated in 2D or 3D) at, A, day 4 and, B, day 7. D, Quantification of GAG produced in NP cells (Tie2 + and Tie2 − ) in 2D or in 3D at day 7. E, Quantification of DNA measured in NP cells (Tie2 + and Tie2 − ) in 2D or in 3D after day 7. F‐I, Quantification of the relative gene expression on NP cells (Tie2 + and Tie2 − ) after culture in 2D or in 3D for 1 week of culture. F, Relative gene expression of homeobox protein NANOG ( NANOG ) in NP cells (Tie2 + and Tie2 − ) in 2D or in 3D. G, Relative gene expression of SRY (sex determining region Y)‐box 2 ( SOX2 ) in NP cells (Tie2 + and Tie2 − ) in 2D or in 3D. H, Relative gene expression of octamer‐binding transcription factor 4 ( OCT4 ) in NP cells (Tie2 + and Tie2 − ) in 2D or in 3D. I, Relative gene expression of angiopoietin‐1 receptor ( TEK ) in NP cells (Tie2 + and Tie2 − ) in 2D or in 3D ( n = 5) for each gene. Data are presented as mean ± SD. Culture of NP cells (Tie2 − ) = blue bars. Culture of NP cells (Tie2 + ) = red bars

Article Snippet: Tie2 + cells were sorted using FACS as previously described., , Freshly isolated NPCs (N = 3) were incubated with an anti‐human Tie2 PE‐conjugated monoclonal mouse antibody (#FAB3131P, clone 83 715, R&D systems) for 30 minutes on ice in 100 μL of FACS buffer ([PBS] with 0.5% bovine serum albumin [BSA] and 1 mM EDTA, all from Sigma‐Aldrich), protected from light.

Techniques: Comparison, Dissection, Marker, Alamar Blue Assay, Produced, Gene Expression, Binding Assay

Journal: Molecular Cancer Research

Article Title: Expression of the Receptor Tyrosine Kinase Tie2 in Neoplastic Glial Cells Is Associated with Integrin β1-Dependent Adhesion to the Extracellular Matrix

doi: 10.1158/1541-7786.mcr-06-0184

Figure Lengend Snippet: FIGURE 2. Tie2 is expressed in human glioma cell lines. A. The presence of Tie2 transcripts is shown in a panel of human glioma cell lines, as assessed by RT-PCR. HUVECs and 293 cells were used as positive and negative controls, respectively. Amplification of glyceraldehyde-3- phosphate dehydrogenase is shown as an internal control. (), PCR reaction without RNA template. B. Immunoblot analyses of Tie2 protein expression in glioma cultures. Tie2 is detected in the membrane fraction of U-87 MG and D54 MG glioma cells but not in U-251 MG and 293 (negative control) cells. HUVEC lysates were used as a positive control.

Article Snippet: Flow Cytometric Analysis For the flow cytometric analysis, tumor-derived cell suspensions (2-5 10 cells) were first treated with FcR blocking reagent (Miltenyi Biotec, Auburn, CA) to block unwanted binding of antibodies to the cells and then incubated with PE-conjugated mouse anti-human Tie2 (83715, R&D Systems), FITC-conjugated mouse anti-human CD31 (WM59), and APC-conjugated mouse anti-human CD34 (BD Biosciences, San Diego, CA) antibodies for 30 min at 4jC.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Control, Western Blot, Expressing, Membrane, Negative Control, Positive Control

Journal: Molecular Cancer Research

Article Title: Expression of the Receptor Tyrosine Kinase Tie2 in Neoplastic Glial Cells Is Associated with Integrin β1-Dependent Adhesion to the Extracellular Matrix

doi: 10.1158/1541-7786.mcr-06-0184

Figure Lengend Snippet: FIGURE 4. Tie2 expression modulates glioma cell adhesion to Ang1. A. Characterization of the Tie2-expressing U251.Tie2 cells. Fluorescence-activated cell sorting analysis of the expression of Tie2 in the surface of U251.vector and the U251.Tie2 stable clones using anti-Tie2 antibody. Fluorescence intensity is expressed in arbitrary units. B. Adhesion of U-251 MG cells to Ang1. Glioma cells (1 105) were plated onto BSA- or Ang1-coated wells. Results are expressed as the fold increase in the adhesion of cells to Ang1 compared with BSA, equal to 1. Experiments were done in triplicate. *, P < 0.001 compared with adhesion to BSA-coated wells. C. Representative experiment of U251.vector and U251.Tie2 (clone no. 3) cell adhesion to BSA- or Ang1-coated wells (magnification, 100). D. Blockage of U251.Tie2 cell adhesion to Ang1 by soluble Tie2-Fc (sTie2Fc). Adhesion of U251.Tie2 cells (clone no. 3, 1 105) to coated Ang1 (10 Ag/mL) was measured in the absence or presence of 50 Ag/mL hFc or sTie2-Fc. Results are expressed as the fold increase in the adhesion of cells to Ang1 compared with BSA, equal to 1. Columns, means from at least three independent experiments, each done in triplicate; bars, SD; *, P < 0.001, compared with hFc-treated U251.Tie2 cells.

Article Snippet: Flow Cytometric Analysis For the flow cytometric analysis, tumor-derived cell suspensions (2-5 10 cells) were first treated with FcR blocking reagent (Miltenyi Biotec, Auburn, CA) to block unwanted binding of antibodies to the cells and then incubated with PE-conjugated mouse anti-human Tie2 (83715, R&D Systems), FITC-conjugated mouse anti-human CD31 (WM59), and APC-conjugated mouse anti-human CD34 (BD Biosciences, San Diego, CA) antibodies for 30 min at 4jC.

Techniques: Expressing, Fluorescence, FACS, Plasmid Preparation, Clone Assay

Journal: Molecular Cancer Research

Article Title: Expression of the Receptor Tyrosine Kinase Tie2 in Neoplastic Glial Cells Is Associated with Integrin β1-Dependent Adhesion to the Extracellular Matrix

doi: 10.1158/1541-7786.mcr-06-0184

Figure Lengend Snippet: FIGURE 5. Treatment with sTie2-Fc or Tie2 siRNA decreases adhesion of Tie2+ glioma cells, U-87 MG, to Ang1. A. Inhibition of U-87 MG cell adhesion to Ang1 by sTie2-Fc. Adhesion of U-87 MG cells (1 105) to Ang1 was measured in the absence or presence of 50 Ag/mL of hFc or sTie2-Fc. Results are expressed as the fold increase in U-87 MG cell adhesion to Ang1 in the presence of sTie2Fc compared with hFc, equal to 1. Experiments were done in triplicate. *, P < 0.05 compared with hFc-treated U-87 MG cells. B. RT-PCR and Western blotting analyses showed down-regulation of Tie2 mRNA and protein levels, respectively, in U-87 MG cells after transfection with Tie2 siRNA, or Tie2 shRNA compared with controls. Glyceraldehyde-3-phosphate dehydrogenase and epidermal growth factor receptor levels are shown as the loading controls. C. Adhesion to Ang1 of U-87 MG cells (1 105) transfected with controls, Tie2 siRNA (50 nmol/L), or Tie2 shRNA and then plated onto 10 Ag/mL of BSA- or Ang1-coated wells. Results are expressed as the fold increase in the adhesion of cells to Ang1 compared with BSA, equal to 1. *, P < 0.05 compared with control siRNA- or shRNA-transfected U-87 MG cells in Ang1-coated wells.

Article Snippet: Flow Cytometric Analysis For the flow cytometric analysis, tumor-derived cell suspensions (2-5 10 cells) were first treated with FcR blocking reagent (Miltenyi Biotec, Auburn, CA) to block unwanted binding of antibodies to the cells and then incubated with PE-conjugated mouse anti-human Tie2 (83715, R&D Systems), FITC-conjugated mouse anti-human CD31 (WM59), and APC-conjugated mouse anti-human CD34 (BD Biosciences, San Diego, CA) antibodies for 30 min at 4jC.

Techniques: Inhibition, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, shRNA, Control

Journal: Molecular Cancer Research

Article Title: Expression of the Receptor Tyrosine Kinase Tie2 in Neoplastic Glial Cells Is Associated with Integrin β1-Dependent Adhesion to the Extracellular Matrix

doi: 10.1158/1541-7786.mcr-06-0184

Figure Lengend Snippet: FIGURE 6. The presence of Tie2 is associated with the expression of integrin h1 and adhesion of glioma cells to ECM. A. Adhesion of U251.Tie2 cells (3 104 cells) to collagen I (Col I), collagen IV (Col IV), laminin (LM), fibronectin (FN), and vitronectin (VN; 10 nmol/L) was measured and compared with the adhesion of U251.vector cells. Results are expressed as the fold increase in the adhesion to every ECM component compared with BSA, equal to 1. *, P < 0.005 compared with U251.vector cells. B. Adhesion of U251.Tie2 cells (1 105 cells) to coatings of collagen I and IV (Col I, Col IV; 10 nmol/L) in the presence of EDTA (10 mmol/L) was measured. Results are represented as the fold increase in the adhesion of cells to ECM component-coated wells compared with BSA, equal to 1. *, P < 0.001 compared with adhesion of cells to ECM-coated wells in the absence of EDTA. C. Molecular modifications related to Tie2 expression. Expression pattern of total and phospho-Tie2, integrins h1 and h4, and total and phospho-FAK in U251.vector and U251.Tie2 cells. Whole cell lysates (WCL) or lysates obtained after Tie2 immunoprecipitation (IP) were assessed by Western blotting. Actin expression is shown as a loading control. D. Adhesion of glioma cells to collagen I (Col I) depended on integrin h1. U-251.Tie2 cells (3 104) were preincubated with anti-integrin h1 (anti-h1) or isotype (IgG) antibodies (10 Ag/mL) for 10 min and plated onto wells coated with BSA or collagen I (10 nmol/L). Results are expressed as the fold increase in cell adhesion to collagen compared with BSA, equal to 1. *, P < 0.001 compared with isotype antibody-treated U-251.Tie2 cells. E. U-251.Tie2 or U-251.vector cells were allowed to attach to chamber slides coated with 50 Ag/mL of collagen I for 3 h at 37jC. Cells were fixed, permeabilized, and incubated with anti-paxillin antibody followed by FITC-conjugated anti-mouse antibody. Arrows, focal adhesion contacts.

Article Snippet: Flow Cytometric Analysis For the flow cytometric analysis, tumor-derived cell suspensions (2-5 10 cells) were first treated with FcR blocking reagent (Miltenyi Biotec, Auburn, CA) to block unwanted binding of antibodies to the cells and then incubated with PE-conjugated mouse anti-human Tie2 (83715, R&D Systems), FITC-conjugated mouse anti-human CD31 (WM59), and APC-conjugated mouse anti-human CD34 (BD Biosciences, San Diego, CA) antibodies for 30 min at 4jC.

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Control, Incubation

Journal: Molecular Cancer Research

Article Title: Expression of the Receptor Tyrosine Kinase Tie2 in Neoplastic Glial Cells Is Associated with Integrin β1-Dependent Adhesion to the Extracellular Matrix

doi: 10.1158/1541-7786.mcr-06-0184

Figure Lengend Snippet: FIGURE 7. Ang1 signals through Tie2 to regulate integrin h1 and adhesion to ECM. A. Ang1 treatment resulted in Tie2 phosphorylation in U-87 MG cells. Tie2-immunoprecipitated lysates (IP) from serum-starved U-87 MG cells untreated or treated with Ang1 (500 ng/mL) for 10 min were analyzed by immunoblotting with anti-Tie2 and phosphotyrosine antibodies. B and C. Ang1 treatment resulted in up-regulation of integrin h1 in U-87 MG cells. B. Lysates from serum-starved U-87 MG cells untreated or treated with Ang1 (500 ng/mL) for the indicated times were analyzed by immunoblotting with anti-integrin h1 antibody. Representative experiment is shown. Actin expression was used as loading control (top). Relative integrin h1 level was quantified from three independent experiments as described in Materials and Methods. Signal intensity for integrin h1 was normalized to that of actin levels of expression for every sample, and graphed as relative to untreated sample (0 h; equal to 100%; bottom). C. Expression levels of integrin h1 in the cell surface were determined by flow cytometric analysis. Isotype IgG was used as negative control (gray filled). D. Knockdown of Tie2 expression inhibited Ang1-induced expression of integrin h1. U-87 MG cells were transfected with Tie2 siRNA or control siRNA (40 nmol/L). Serum-starved U-87 MG cells were stimulated with Ang1 (500 ng/mL) for 3 h. Left, cell lysates were analyzed by immunoblotting with anti-integrin h1 antibody. Representative experiment is shown. Actin expression is shown as the loading control. Right, relative protein levels were quantified as described in Materials and Methods. Signal intensity for integrin h1 was normalized to that of actin levels of expression for every sample, and graphed as relative to mock-treated samples (equal to 100%). E. Knockdown of Tie2 expression inhibited Ang1-mediated adhesion to collagen I (Col I) and collagen IV (Col IV). U-87 MG cells were transfected with Tie2 siRNA or control siRNA (40 nmol/L). Serum-starved U-87 MG cells were stimulated with Ang1 (500 ng/mL) for 3 h, and then 3 105 cells were plated on collagen I- or collagen IV- coated wells (10 nmol/L). Results are expressed as the fold increase in cell adhesion to collagen compared with BSA, equal to 1. *, P < 0.005; **, P < 0.05 compared with control siRNA-treated cells.

Article Snippet: Flow Cytometric Analysis For the flow cytometric analysis, tumor-derived cell suspensions (2-5 10 cells) were first treated with FcR blocking reagent (Miltenyi Biotec, Auburn, CA) to block unwanted binding of antibodies to the cells and then incubated with PE-conjugated mouse anti-human Tie2 (83715, R&D Systems), FITC-conjugated mouse anti-human CD31 (WM59), and APC-conjugated mouse anti-human CD34 (BD Biosciences, San Diego, CA) antibodies for 30 min at 4jC.

Techniques: Phospho-proteomics, Immunoprecipitation, Western Blot, Expressing, Control, Negative Control, Knockdown, Transfection