Journal: Journal of Clinical Medicine

Article Title: Compartment-Specific Differences in the Activation of Monocyte Subpopulations Are Not Affected by Nitric Oxide and Glucocorticoid Treatment in a Model of Resuscitated Porcine Endotoxemic Shock

doi: 10.3390/jcm11092641

Figure Lengend Snippet: Endotoxin-induced changes in the circulating cell counts and phenotypes. Time-course changes in: ( A ) White blood count (WBC), ( B ) Circulating mononuclear cells (MNCs), and ( C ) CD3 + T-cells. ( D ) Representative dot plots of the flow cytometric analysis of blood monocytes; first, all cells were gated, and then the gate for the MNCs was applied and this population was analyzed for the expression of CD163 and CD14 before the infusion of endotoxin, 4 h and 20 h after the start of endotoxemia. The frequency of: ( E ) CD14 + CD163 − monocytes, ( F ) CD14 + CD163 + monocytes, and ( G ) CD14 − CD163 + monocytes is shown. Mean values ± SD are presented. Baseline, T4 h T8 h n = 9/group, T20 h n = 6/group. The statistical significance of differences between values at different timepoints for each group: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The statistical significance of differences between groups at given timepoints: ## p < 0.01. FSC forward scatter, SSC side scatter.

Article Snippet: The following antibodies for immunophenotyping of the monocytes were used: anti-CD14-PE.Cy7 (clone M5E2, BioLegend, San Diego, CA, USA), anti-CD163 (2A10/11, Bio-Rad (AbD Serotec), Hercules, CA, USA) labeled with Zenon AlexaFluor 647 mouse IgG1 (Thermofisher), and anti-SLA-DR-FITC (2E9/13, Bio-Rad (AbD Serotec)).

Techniques: Expressing

Journal: Journal of Clinical Medicine

Article Title: Compartment-Specific Differences in the Activation of Monocyte Subpopulations Are Not Affected by Nitric Oxide and Glucocorticoid Treatment in a Model of Resuscitated Porcine Endotoxemic Shock

doi: 10.3390/jcm11092641

Figure Lengend Snippet: Effects of endotoxemia on the activation status of circulating monocytes. The kinetics of SLA-DR expression on subpopulations of circulating blood monocytes with representative histograms from flow cytometry analysis is shown: ( A ) CD14 + CD163 − monocytes, ( B ) CD14 + CD163 + monocytes, and ( C ) CD14 − CD163 + monocytes. The mitochondrial membrane potential (Δφ) was analyzed using JC-1 staining followed by flow cytometric analysis on: ( D ) CD14 + CD163 + monocytes, ( E ) CD14 + CD163 + monocytes, and ( F ) CD14 + CD163 − monocytes. Mean values ±SD are presented. Baseline, T4 h T8 h n = 9/group, T20 h n = 6/group. The statistical significance of differences between values at different timepoints for each group: ** p < 0.01, *** p < 0.001, **** p < 0.0001. SLA-DR swine leukocyte antigen-DR.

Article Snippet: The following antibodies for immunophenotyping of the monocytes were used: anti-CD14-PE.Cy7 (clone M5E2, BioLegend, San Diego, CA, USA), anti-CD163 (2A10/11, Bio-Rad (AbD Serotec), Hercules, CA, USA) labeled with Zenon AlexaFluor 647 mouse IgG1 (Thermofisher), and anti-SLA-DR-FITC (2E9/13, Bio-Rad (AbD Serotec)).

Techniques: Activation Assay, Expressing, Flow Cytometry, Staining

Journal: Journal of Clinical Medicine

Article Title: Compartment-Specific Differences in the Activation of Monocyte Subpopulations Are Not Affected by Nitric Oxide and Glucocorticoid Treatment in a Model of Resuscitated Porcine Endotoxemic Shock

doi: 10.3390/jcm11092641

Figure Lengend Snippet: Compartment-specific changes in the activation status of monocytes following 20 h of endotoxemic shock. The expression of SLA-DR antigen was evaluated 20 h after the onset of endotoxemic shock in cells from the bronchoalveolar lavage fluid (BAL), peripheral blood (PB), and bone marrow (BM). The SLA-DR antigen was analyzed in: ( A ) CD14 + CD163 − monocytes, ( B ) CD14 + CD163 + monocytes, and ( C ) CD14 − CD163 + monocytes. The mitochondria membrane potential (Δφ) was also analyzed in these three compartments in: ( D ) CD14 + CD163 − monocytes, ( E ) CD14 + CD163 + monocytes, and ( F ) CD14 − CD163 + monocytes. Mean values ± SD are presented. n = 6/group. The statistical significance of differences between values for BAL vs. PB or BM in each experimental group: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Significant differences between PB and BM §§§§ p < 0.0001. No differences were noted between control and treated group.

Article Snippet: The following antibodies for immunophenotyping of the monocytes were used: anti-CD14-PE.Cy7 (clone M5E2, BioLegend, San Diego, CA, USA), anti-CD163 (2A10/11, Bio-Rad (AbD Serotec), Hercules, CA, USA) labeled with Zenon AlexaFluor 647 mouse IgG1 (Thermofisher), and anti-SLA-DR-FITC (2E9/13, Bio-Rad (AbD Serotec)).

Techniques: Activation Assay, Expressing

Journal: Veterinary Research

Article Title: Porcine mononuclear phagocyte subpopulations in the lung, blood and bone marrow: dynamics during inflammation induced by Actinobacillus pleuropneumoniae

doi: 10.1051/vetres/2010035

Figure Lengend Snippet: Primary mouse anti-porcine cell surface marker antibodies used in the experiment.

Article Snippet: CD163 , 2A10/11 , IgG1 , Serotec , MCA2311 , subset of mo/mf, DC.

Techniques: Marker, Expressing

Journal: The Journal of General Virology

Article Title: Molecular cloning of porcine Siglec-3, Siglec-5 and Siglec-10, and identification of Siglec-10 as an alternative receptor for porcine reproductive and respiratory syndrome virus (PRRSV)

doi: 10.1099/jgv.0.000859

Figure Lengend Snippet: Expression analysis of Siglec-3, Siglec-5 and Siglec-10 using IFA and Western blotting. (a) PK-15 cells were transfected with Siglec-3-, Siglec-5- and Siglec-10-encoding vector. Twenty-four hours post-transfection, the cells were fixed with 4 % PF and permeabilized with Triton X-100 for cytoplasmic staining, or not permeabilized for surface staining. IFA was performed using V5-specific antibody (Siglec; green) and Hoechst 33 342 (nuclei; blue). Scale bar: 25 µm (b) Western blot identification. HEK-293T cells were transfected with Siglec-3, Siglec-5 and Siglec-10. Twenty-four hours post-transfection, the cells were collected and lysed with lysis buffer. Afterwards, cellular lysates were treated or mock-treated with sialidase, EndoH or PNGase F and analysed by SDS-PAGE and Western blot. Primary antibody V5-specific mAb (GenScript; A00641) and secondary peroxidase-labelled goat anti-mouse IgG antibodies (Dako) were used for immunoblotting. For the detection of tubulin, an HRP-conjugated anti-alpha tubulin monoclonal antibody (Abcam; ab40742) was used.

Article Snippet: For the visualization of CD163, either a mouse monoclonal antibody (2A10/11, IgG1; AbD Serotec, Dusseldorf, Germany) or a goat polyclonal antibody (R and D Systems, Minneapolis) with appropriate secondary conjugates were used.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Staining, Lysis, SDS Page

Journal: The Journal of General Virology

Article Title: Molecular cloning of porcine Siglec-3, Siglec-5 and Siglec-10, and identification of Siglec-10 as an alternative receptor for porcine reproductive and respiratory syndrome virus (PRRSV)

doi: 10.1099/jgv.0.000859

Figure Lengend Snippet: Virus production for the different transfected PK-15 cell groups 24 h after infection. PK-15 cells were transiently transfected with a pCD163-encoding vector in combination with a Siglec-1, Siglec-3, Siglec-5, Siglec-10 or control vector, and 48 h after transfection the cells were treated or not treated with sialidase for 1 h and inoculated with PRRSV LV or MN-184 for 1 h. Twenty-four hours post-infection, the cells were fixed and stained for infection and expression of the different Siglecs and CD163. (a) Immunofluorescence staining of infected cells with mAb 13E2 (against PRRSV nucleocapsid protein; green) and Hoechst 33 342 (nuclei; blue). The absolute number of infected cells for each condition was determined and displayed in the images as the mean ± SEM of three independent experiments. Scale bar: 50 µm. (b) Expression analysis of the different Siglecs using fluorescence microscopy. PK-15 cells were fixed, permeabilized and stained with V5-specific mAb (green) and Hoechst 33 342 (nuclei; blue). The absolute number of transfected cells was determined for each condition and is displayed in the images as the mean ± SEM of three independent experiments. Scale bar: 50 µm. (c) To evaluate virus production, the cell supernatants collected at 24 h p.i. were titrated and the results are displayed in the graphs. The CD163/Siglec double-transfected groups that were significantly different from the CD163 single-transfected group are represented as * P <0.05; ** P <0.01 and *** P <0.001.

Article Snippet: For the visualization of CD163, either a mouse monoclonal antibody (2A10/11, IgG1; AbD Serotec, Dusseldorf, Germany) or a goat polyclonal antibody (R and D Systems, Minneapolis) with appropriate secondary conjugates were used.

Techniques: Virus, Transfection, Infection, Plasmid Preparation, Control, Staining, Expressing, Immunofluorescence, Fluorescence, Microscopy

Journal: The Journal of General Virology

Article Title: Molecular cloning of porcine Siglec-3, Siglec-5 and Siglec-10, and identification of Siglec-10 as an alternative receptor for porcine reproductive and respiratory syndrome virus (PRRSV)

doi: 10.1099/jgv.0.000859

Figure Lengend Snippet: Porcine Siglec-10 mediates the endocytosis of PRRSV. (a) Immunofluorescence staining of Siglec-10 in stably transfected PK-15 S10+ cells. PK-15 S10+ cells were fixed with 4 % PF, and the cells were permeabilized (cytoplasmic staining) or not permeablized (surface staining) with 0.1 % Triton X-100 and stained with 1G10 mAb against Siglec-10 (green) and Hoechst 33 342 (nuclei; blue). Scale bar: 25 µm. (b) Attachment and internalization of PRRSV in PK-15 S10+ cells. PK-15 expressing Siglec-10 or normal PK-15 cells were incubated with purified PRRSV LV for 1 h at 4 °C or 37 °C, allowing binding and internalization, respectively. After washing, the cells were fixed and stained with Hoechst 33 342 (nuclei; blue) and mAb 13E2 (PRRSV nucleocapsid protein; green), and analysed by confocal microscopy. Scale bar: 25 µm (c) CHO and PK-15 cells were transiently transfected with wild-type Siglec-10 or the Siglec-10 R119E mutant, and a virus internalization assay was performed 48 h post-transfection. Double staining for Siglec-10/Siglec-10 R119E (red) and co-localized PRRSV particles (13E2; green) was performed and analysed by confocal microscopy. Scale bar: 25 µm. (d) Quantification of PRRSV internalization in CHO and PK-15 cells for three independent experiments.

Article Snippet: For the visualization of CD163, either a mouse monoclonal antibody (2A10/11, IgG1; AbD Serotec, Dusseldorf, Germany) or a goat polyclonal antibody (R and D Systems, Minneapolis) with appropriate secondary conjugates were used.

Techniques: Immunofluorescence, Staining, Stable Transfection, Transfection, Expressing, Incubation, Purification, Binding Assay, Confocal Microscopy, Mutagenesis, Virus, Double Staining

Journal: Stem Cell Reviews and Reports

Article Title: The Intrapericardial Delivery of Extracellular Vesicles from Cardiosphere-Derived Cells Stimulates M2 Polarization during the Acute Phase of Porcine Myocardial Infarction

doi: 10.1007/s12015-019-09926-y

Figure Lengend Snippet: Monocyte populations in peripheral blood. Blood samples were collected in EDTA containing tubes before acute myocardial infarction model creation (Basal), 72 h after (Post-AMI) and 24 h after the treatment (Post-therapy). Monocyte count was performed in an automated hematology analyzer and its phenotype characterization was evaluated by flow cytometry, defining circulating M2 monocytes as CD14 + CD163+. Normality was assessed using a Shapiro-Wilk test. Paired comparisons of the AMI model ( a )(n = 15) and paired comparisons of the administered therapies ( b ) (n = 5) were performed using a Student t-test for parametric data or a Wilcoxon signed rank test with the Yates continuity correction for non-parametric variables. Graphs show the mean ± SD of cell populations. **p ≤ 0.01

Article Snippet: Cells were labelleded with fluorescent-dye anti-porcine monoclonal antibodies for the following surface molecules: CD4 (clone 74–12-4, BD Pharmingen, CA, USA), CD8α (clone 76–2-11, BD Pharmingen, CA, USA), CD14 (clone TÜK4, Bio-Rad, CA, USA), CD16 (clone G7, Bio-Rad, CA, USA), CD27 (clone B30C7, Bio-Rad, CA, USA), CD45RA (clone MIL13, Bio-Rad, CA, USA), CD107a (clone 4E9/11, Bio-Rad, CA, USA), CD163 (clone 2A10/11, BD Pharmingen, CA, USA), and SLA-II (clone 2E9/13, Bio-Rad, CA, USA).

Techniques: Flow Cytometry