Journal: Glia

Article Title: The Transcription Factor p53 Influences Microglial Activation Phenotype

doi: 10.1002/glia.21178

Figure Lengend Snippet: Separate populations of microglia demonstrate immunoreactivity for a marker of alternatively activated and deactivated macrophages, CD163 or p53 activation in HAND cases. A) Human cortical tissue sections immunolabeled for p53 (brown) and CD68 (purple) reveal that a portion of microglia are immunoreactive for both markers. Open arrows point to double labeled cells and closed arrows identify CD68 single labeled cells. B) A section adjacent to the one in A is shown labeled with antibodies to p53 (brown) and CD163 (purple). Closed arrows point to CD163 labeled microglia. C) Quantification of double labeled microglia in adjacent sections from twelve HAND patients (*=p<0.0001 using a paired t-test).

Article Snippet: Co-labeling for p53 and CD163 was performed by adapting a previously published protocol ( Fischer-Smith et al. 2008 ) using a monoclonal antibody to CD163 (Vector 1:50) and co-labeling with antibodies to p53 using the same method as that developed to double label for p53 and CD68.

Techniques: Marker, Activation Assay, Immunolabeling, Labeling

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 1. Changes of cytokine concentration in THP-1 macrophages challenged with a single LPS stimulation and a CD163 antibody. Quantification for IL-10 (A), TGF-β (B), IL-1 β (C), TNF- α (D), MCP-1 (E), and IL-6 (F) protein concentration in THP-1 macrophages with a single LPS stimulation and incubated with the anti-CD163 antibody (RM3/1) or its isotype control (Iso) for 24 h (72 h after the addition of LPS). N = 6–10 per group. *P < 0.05, vs. LPS, #P < 0.05, vs. isotype control by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Concentration Assay, Protein Concentration, Incubation, Control

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 2. Changes of cytokine concentration in primary human macrophages challenged with a single LPS stimulation and a CD163 antibody. Quantification for IL-10 (A), TGF-β (B), IL-1 β (C), TNF-α (D), MCP-1 (E), and IL-6 (F) protein concentration in primary human macrophages with a single LPS stimulation and incubated with the anti-CD163 antibody (RM3/1) or its isotype control (Iso) for 24 h (72 h after the addition of LPS). N = 6–10 per group. #P < 0.05, vs. isotype control by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Concentration Assay, Protein Concentration, Incubation, Control

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 3. CD163 mRNA induction in THP-1 macrophages challenged with a single LPS stimulation. Quantification for CD163 mRNA induction using Man-PEI complexed with either a plasmid encoding for CD163 gene (pCD163) or the empty vector (pEmpty) in LPS-stimulated THP-1 macrophages at 24, 48, 72 and 96 h after transfection. The expression of CD163 mRNA was normalized to the respective levels of β-actin expression in each group and then calculated as fold change against the expression of the control group (Man-PEI) at each time point. N = 5–6 per group. *P < 0.05 vs. pEmpty by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Plasmid Preparation, Transfection, Expressing, Control

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 4. CD163 protein induction in THP-1 macrophages challenged with a single LPS stimulation. Microscopic images of nuclear staining using DAPI (blue), CD163 protein (red) and mannose receptor (CD206, green) in LPS-stimulated THP-1 macrophages transfected with Man-PEI complexed with an empty vector (pEmpty), a plasmid encoding for CD163 gene (pCD163) or the Man-PEI nanoparticle alone (A). Quantification of the relative average fluorescence intensity of CD163 (B) or mannose receptor (CD206, C) in LPS-stimulated THP-1 macrophages from 48 to 96 h after transfection. The quantification of the relative average fluorescence intensity was normalized to the respective levels in the Man-PEI group, which was assigned a value equal to 1. N = 3 per group. *P < 0.05 vs. pEmpty by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Staining, Transfection, Plasmid Preparation

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 5. CD163 protein induction in THP-1 macrophages challenged with a single LPS stimulation. Representative Western blot images of CD163 and Beta-Actin proteins in LPS-stimulated THP-1 macrophages transfected with Man-PEI complexed with an empty vector (pEmp or pEmpty), a plasmid encoding for CD163 gene (pCD163) or the Man-PEI nanoparticle alone (A). Quantification of the relative expression (band density) of CD163 in LPS-stimulated THP-1 macrophages from 48 h after transfection (B). The quantification of the relative density was normalized to the respective levels in the pEmpty group, which was assigned a value equal to 1. N = 10 for pCD163 or pEmpty group. *P < 0.05 vs. pEmpty by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 6. Changes in cytokine expression in CD163-overexpressing THP-1 macrophages challenged with a single LPS stimulation. Quantification for IL-10 (A), IL-1ra (B), TGF-β (C), TNF-α (D), MCP-1 (E), IL-1β (F), and IL-6 (G) protein concentration in THP-1 macrophages with a single LPS stimulation and transfected with either a plasmid encoding for CD163 gene (pCD163) or the empty vector (pEmpty) at 24, 48, 72 and 96 h after transfection. The concentration of each cytokine/chemokine was normalized to the control group (Man-PEI), which was assigned a value equal to 1. N = 4–9 per group. *P < 0.05, vs. pEmpty by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Expressing, Protein Concentration, Transfection, Plasmid Preparation, Concentration Assay, Control

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 7. CD163 mRNA induction in THP-1 macrophages challenged with a double LPS stimulation. Quantification for CD163 mRNA using Man-PEI complexed with either a plasmid encoding for CD163 gene (pCD163) or the empty vector (pEmpty) in THP-1 macrophages with a double LPS stimulation at 4 and 24 h after the second stimulus (+4 and +24, respectively). The expression of CD163 mRNA was normalized to the respective levels of β-actin expression in each group and then calculated as fold change against the expression of the control group (Man-PEI) at each time point, which was assigned a value equal to 1. N = 4–6 per group. *P < 0.05 against the pEmpty transfected group by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Plasmid Preparation, Expressing, Control, Transfection

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 8. Changes of cytokine expression in CD163-overexpressing THP-1 macrophages challenged with a double LPS stimulation. Quantification for IL-10 (A), IL-1ra (B), TGF-β (C), TNF-α (D), MCP-1 (E), IL-1β (F), and IL-6 (G) protein concentration in THP-1 macrophages with a double LPS stimulation and transfected with either a plasmid encoding for CD163 gene (pCD163) or the empty vector (pEmpty) at 4 and 24 h after the second stimulus (+4 and +24, respectively). The concentration of each molecule was normalized to the control group (Man-PEI), which was assigned a value equal to 1. N = 5–13 per group. *P < 0.05, vs. pEmpty by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Expressing, Protein Concentration, Transfection, Plasmid Preparation, Concentration Assay, Control

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 9. Changes of cytokine expression in CD163-overexpressing THP-1 macrophages challenged with a double LPS stimulation and a CD163 antibody. Quantification for IL-10 (A) and IL- 1ra (B) protein concentration in THP-1 macrophages transfected with either a plasmid encoding for CD163 gene (pCD163) or the empty vector (pEmpty) and incubated with either anti- CD163 antibody (RM3/1) or its isotype control antibody at 4 (for IL-10) and 24 (for IL-1ra) h after a second LPS stimulus. The protein concentration of each group was normalized to the control group (pEmpty), which was assigned a value equal to 1. N = 5–12 per group. *P < 0.05 using Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Expressing, Protein Concentration, Transfection, Plasmid Preparation, Incubation, Control

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 10. CD163 mRNA induction in primary human macrophages challenged with a single LPS stimulation. Quantification for CD163 mRNA induction using Man-PEI complexed with either a plasmid encoding for CD163 gene (pCD163) or the empty vector (pEmpty) in primary human macrophages with a single stimulation of LPS at 48 and 96 h after transfection. The expression of CD163 mRNA was normalized to the respective levels of β-actin expression in each group, and then calculated as fold change against the expression of the pEmpty group at each time point, which was assigned a value equal to 1. N = 6–9 per group. *P < 0.05 against the pEmpty group by Student's t- test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Plasmid Preparation, Transfection, Expressing

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 11. Changes of cytokine expression in CD163-overexpressing primary human macrophages challenged with a single LPS stimulation. Quantification for IL-10 (A), IL-1ra (B), TGF-β (C), TNF-α (D), MCP-1 (E), IL-1β (F), and IL-6 (G) protein concentration in primary human macrophages with a single LPS stimulation and transfected with either a plasmid encoding for CD163 gene (pCD163) or the empty vector (pEmpty) at 48 and 96 h after transfection. The concentration of each molecule was normalized to the control group (pEmpty), which was assigned a value equal to 1. N = 5–8 per group. *P < 0.05 vs. pEmpty by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Expressing, Protein Concentration, Transfection, Plasmid Preparation, Concentration Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Multiple Immunostainings with Different Epitope Retrievals—The FOLGAS Protocol

doi: 10.3390/ijms23010223

Figure Lengend Snippet: Secondary antibodies, chromogens, fluorophores and metals. Except DAPI, the fluorophores not directly coupled to antibodies are linked to streptavidin for binding of biotinylated antibodies. Abbreviations: RTU: ready to use, AP: alkaline phosphatase, HRP: horseradish peroxidase. Tb: Terbium, Er: Erbium.

Article Snippet: , 170Er (conjugated to CD163 clone EDHu-1) , , - , Fluidigm (SKU 201300).

Techniques: Binding Assay

Journal: International Journal of Oncology

Article Title: Effect of macrophages on breast cancer cell proliferation, and on expression of hormone receptors, uPAR and HER-2

doi: 10.3892/ijo.2017.3996

Figure Lengend Snippet: Immunohistochemical staining of 17 breast cancer biopsies.

Article Snippet: The sections were incubated for 30 min with either of the following primary antibodies: Monoclonal rabbit anti-human estrogen receptor (ER)α (clone EP1, ready-to-use), monoclonal mouse anti-human progesterone receptor (PR; clone PgR 636, ready-to-use), monoclonal mouse anti-human-CD68 antibody (clone Kp1, ready-to-use), HercepTestTM polyclonal rabbit anti-human HER2, monoclonal mouse anti-human estrogen receptor β1 (clone PPG5/10, dilution 1:40), monoclonal mouse anti-human Ki-67 (clone MIB1, ready-to-use), polyclonal rabbit anti-human MMP-9 (1:50), monoclonal mouse anti-human uPAR (clone R4, 1:50) (all from Dako) and monoclonal mouse anti-human-CD163 antibody (clone 10D6, 1:200, Novocastra, Leica Microsystems, Newcastle, UK).

Techniques: Immunohistochemical staining, Staining

Journal: International Journal of Oncology

Article Title: Effect of macrophages on breast cancer cell proliferation, and on expression of hormone receptors, uPAR and HER-2

doi: 10.3892/ijo.2017.3996

Figure Lengend Snippet: Immunohistochemical staining of CD68, CD163, ERβ1 and uPAR in human, breast cancer biopsies. Representative images of CD68 (A) score 1, (B) score 2, (C) score 3; CD163 (D) score 1, (E) score 2, (F) score 3; ERβ1 (G) 30% positive tumor cells, (H) 50% positive tumor cells (I) 100% positive tumor cells; uPAR (J) score 1, (K) score 2, (L) score 3 (×400 magnification, calibration bar is 50 µ m in all micrographs).

Article Snippet: The sections were incubated for 30 min with either of the following primary antibodies: Monoclonal rabbit anti-human estrogen receptor (ER)α (clone EP1, ready-to-use), monoclonal mouse anti-human progesterone receptor (PR; clone PgR 636, ready-to-use), monoclonal mouse anti-human-CD68 antibody (clone Kp1, ready-to-use), HercepTestTM polyclonal rabbit anti-human HER2, monoclonal mouse anti-human estrogen receptor β1 (clone PPG5/10, dilution 1:40), monoclonal mouse anti-human Ki-67 (clone MIB1, ready-to-use), polyclonal rabbit anti-human MMP-9 (1:50), monoclonal mouse anti-human uPAR (clone R4, 1:50) (all from Dako) and monoclonal mouse anti-human-CD163 antibody (clone 10D6, 1:200, Novocastra, Leica Microsystems, Newcastle, UK).

Techniques: Immunohistochemical staining, Staining

Journal: PLoS Pathogens

Article Title: Precision engineering for PRRSV resistance in pigs: Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function

doi: 10.1371/journal.ppat.1006206

Figure Lengend Snippet: Peripheral blood monocytes were isolated from the blood of the wild type (red), heterozygous (blue), and ΔSRCR5 (green) animals. Following cultivation in the presence of recombinant human CSF1 (rhCSF1) for seven days PMMs were analyzed by FACS. A) Co-staining with CD14-FITC and CD16-PE antibodies recognizing the native structure of the proteins (colored contour plots; red wild type, blue heterozygous, green ΔSRCR5) relative to isotype controls (grey). B) Co-staining with CD169-FITC and CD172a-PE antibodies recognizing the native structure of the proteins (colored contour plots) relative to isotype controls (grey). C) Co-staining with SWC9 (CD203a)-FITC and CD151-RPE antibodies recognizing the native structure of the proteins (colored contour plots) relative to isotype controls (grey). D) Staining against the native structure of surface expressed CD163 (colored) relative to an isotype control staining (grey).

Article Snippet: Cells were stained with antibodies targeting either mouse anti pig CD14 (AbD Serotec, MGA1273F, 1:50) and mouse anti pig CD16 (AbD Serotec, MCA2311PE, 1:200), mouse anti pig CD169 (AbD Serotec, MCA2316F, 1:50) and mouse anti pig CD172a (SoutherBiotech, 4525–09, 1:400), mouse anti human CD151 (AbD Serotec, MCA1856PE, 1:50) and mouse anti pig SWC9(CD203a) (AbD Serotec, MCA1973F, 1:50), mouse anti pig CD163 (AbD Serotec, MCA2311PE, 1:50), or mouse IgG1 or an IgG2b negative control (AbD Serotec, MCA928PE,MCA691F, or Sigma, F6397; same concentration as primary Ab).

Techniques: Isolation, Recombinant, Staining, Control