rabbit polyclonal anti human cd163  (Proteintech)

Journal: Acta biomaterialia

Article Title: A subtype specific probe for targeted magnetic resonance imaging of M2 tumor-associated macrophages in brain tumors.

doi: 10.1016/j.actbio.2025.01.003

Figure Lengend Snippet: Fig. 6. Immunohistochemical and FACS validations of M2pep-uIONP targeting M2 TAMs. Confocal microscopic images of tumor tissues collected from mice at 24 h after i.v. injection of probes show (A) Cy7-M2pep-uIONP were highly co-localizing with CD68- and CD163-double positive M2 TAM, revealing the targeting specificity. In comparison, the non-targeted control probe Cy7-scM2pep-uIONP and Cy7-Ferumoxytol not only exhibited much less tumoral accumulation, but also were not co-localized with M2 TAM. Scale bars: 37 µm. (B) Flow cytometric zebra plots and (C) the corresponding quantification of CD68- and CD163-double positive M2 TAM targeted by FITC-M2pep-uIONP, FITC-scM2pep-uIONP, and FITC-Ferumoxytol. The gating strategy involves sorting CD163+ M2 TAM population in live cells dissociated from mouse cerebrums by gating on CD68+ pan-macrophage populations, followed by further gating to assess the FITC-labeled nanoparticles.

Article Snippet: Rabbit polyclonal anti-CD163 antibody was purchased from Boster Biological Technology (Pleasanton, CA, USA).

Techniques: Immunohistochemical staining, Injection, Comparison, Control, Labeling

Journal: iScience

Article Title: A single-cell landscape of pre- and post-menopausal high-grade serous ovarian cancer ascites

doi: 10.1016/j.isci.2023.107712

Figure Lengend Snippet: Myeloid-derived cell components in pre- and post-menopausal HGSOC ascites (A) UMAP plot showing the subtypes of myeloid-derived cells derived from HGSOC ascites. Each cluster is color-coded according to cell type. Cluster annotations are indicated in the figure. (B) UMAP plot showing HGSOC ascites myeloid cells, clustered and color-coded, according to patient. Data are represented as mean ± SEM. (C) UMAP plot, showing pre- and post-menopausal myeloid cells by color. (D) Heatmap showing the expression of marker genes in each subtype of myeloid-derived cells. (E) Histogram indicating the proportion of myeloid subgroups in ascites of pre- and post-menopausal patients. (F and G) Representative images of IF staining in Formalin-fixed paraffin-embedded (FFPE) ascites cells, indicating CD68 + and CD163+ cells (F), and SPP1+ CD163+ cells (G) in paired pre- and post-menopausal ascites. Scale bar, 10 μm. Bar chart based on IF staining results shows the relative fluorescence intensity of CD163, CD68, and SPP1 in cells. Data are represented as mean ± SEM. All statistical analyses were conducted using paired t-test. n represents the number of cells. (H) Gene ontology (GO) analysis of pre- (green) and post-menopausal (blue) myeloid cells. (I) The heatmap indicating the gene expression in the myeloid cell subtypes of pre- and post-menopausal HGSOC ascites.

Article Snippet: The following antibodies were used: Monoclonal mouse anti-human CD68 (1:1000, 14-0688-82, Invitrogen), Polyclonal rabbit anti-human CD163 (1:100, PA5-109327, Invitrogen), Monoclonal mouse anti-human Osteopontin (SPP1) (1:500, MA5-17180, Invitrogen), Alexa Fluor 546 cross-adsorbed goat anti-mouse (1:100, A-11003, Invitrogen), Alexa Fluor 488-conjugated goat anti-rabbit (1:100, ab150077, Abcam).

Techniques: Derivative Assay, Expressing, Marker, Staining, Formalin-fixed Paraffin-Embedded, Fluorescence

Journal: iScience

Article Title: A single-cell landscape of pre- and post-menopausal high-grade serous ovarian cancer ascites

doi: 10.1016/j.isci.2023.107712

Figure Lengend Snippet:

Article Snippet: The following antibodies were used: Monoclonal mouse anti-human CD68 (1:1000, 14-0688-82, Invitrogen), Polyclonal rabbit anti-human CD163 (1:100, PA5-109327, Invitrogen), Monoclonal mouse anti-human Osteopontin (SPP1) (1:500, MA5-17180, Invitrogen), Alexa Fluor 546 cross-adsorbed goat anti-mouse (1:100, A-11003, Invitrogen), Alexa Fluor 488-conjugated goat anti-rabbit (1:100, ab150077, Abcam).

Techniques: Suspension, Plasmid Preparation, Expressing, Software

Journal: The Journal of Biological Chemistry

Article Title: The Staphylococcus aureus Protein IsdH Inhibits Host Hemoglobin Scavenging to Promote Heme Acquisition by the Pathogen *

doi: 10.1074/jbc.M116.755934

Figure Lengend Snippet: SPR analysis of CD163-mediated binding of Hb-Hp in presence of IsdH. A and B, sensorgrams displaying binding of Hb-Hp1-1 (A) and Hb-Hp2-2 (B) to immobilized CD163 in presence of 1.5 molar equivalent of IsdHN1, IsdHN2N3, and IsdHN1N2N3 or running buffer as control. C and D, relative plateau response of immobilized CD163 binding of Hb-Hp1-1 (C) and Hb-Hp2-2 (D) are plotted against the indicated molar ratio of IsdHN1 (○), IsdHN2N3 (▾), or IsdHN1N2N3(●). Reproduction of the SPR experiments was ensured by preparing two individual chips with two individual flow cells with either Hp1-1 or Hp2-2. On both chips triplicate repeats of triplicate runs of each sample was performed. The shown data are representative sensorgrams (A and B) and representative results from triplicate experiments to a single flow cell (C and D).

Article Snippet: Polyclonal rabbit anti-human CD163 IgG (Dako), earlier shown to specifically inhibit Hb-Hp uptake by CD163 ( 37 ), and irrelevant polyclonal rabbit anti-human IgG (Dako) were included as inhibition controls, both at a concentration of 50 μg/ml.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The Staphylococcus aureus Protein IsdH Inhibits Host Hemoglobin Scavenging to Promote Heme Acquisition by the Pathogen *

doi: 10.1074/jbc.M116.755934

Figure Lengend Snippet: Analysis of cellular uptake of Hb-Hp2-2 by CD163-expressing cells. A, confocal microscopy images of endocytotic Hb-Hp uptake by CD163-expressing cells. CHO CD163 cells were incubated with 46 μg/ml Atto-488-labeled Hb-Hp2-2 and IsdHN1, IsdHN2N3, or IsdHN1N2N3 in 5-fold molar excess over Hb-Hp. Atto-488-labeled Hb-Hp2-2 is shown in green, DAPI staining of nuclei is shown in blue, and CD163 surface immunostaining (Alexa 647) is shown in red. Mock transfected CHO cells (right) were included as a negative control. B, dose-response inhibition of endocytotic uptake of Atto-488 labeled Hb-Hp2-2 by CHO CD163 cells in presences of increasing molar excess of IsdHN1 (○), IsdHN2N3 (■), or IsdHN1N2N3 (▴). Fluorescence signals were detected by flow cytometry and displayed as MFI. Background uptake in mock transfected CHO cells is indicated with a dashed line. The data are represented as means ± S.D. (n = 3) from one representative experiment of three.

Article Snippet: Polyclonal rabbit anti-human CD163 IgG (Dako), earlier shown to specifically inhibit Hb-Hp uptake by CD163 ( 37 ), and irrelevant polyclonal rabbit anti-human IgG (Dako) were included as inhibition controls, both at a concentration of 50 μg/ml.

Techniques: Expressing, Confocal Microscopy, Incubation, Labeling, Staining, Immunostaining, Transfection, Negative Control, Inhibition, Fluorescence, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: The Staphylococcus aureus Protein IsdH Inhibits Host Hemoglobin Scavenging to Promote Heme Acquisition by the Pathogen *

doi: 10.1074/jbc.M116.755934

Figure Lengend Snippet: Flow cytometric analysis of uptake of Hb-Hp2-2 by dexamethasone-treated monocytes. A, adherent 3-day-old monocytes treated with 2.5 × 10−7 m dexamethasone were incubated with 23 μg/ml Atto-488-labeled Hb-Hp2-2 and IsdHN1, IsdHN2N3, or IsdHN1N2N3 in 1-, 5-, and 20-fold molar excess over Hb-Hp. B, a polyclonal rabbit anti-human CD163 IgG (50 μg/ml) and an irrelevant polyclonal rabbit anti-human antibody (50 μg/ml) were used as positive and negative inhibition controls respectively. The data from one representative experiment of three are shown.

Article Snippet: Polyclonal rabbit anti-human CD163 IgG (Dako), earlier shown to specifically inhibit Hb-Hp uptake by CD163 ( 37 ), and irrelevant polyclonal rabbit anti-human IgG (Dako) were included as inhibition controls, both at a concentration of 50 μg/ml.

Techniques: Incubation, Labeling, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: The Staphylococcus aureus Protein IsdH Inhibits Host Hemoglobin Scavenging to Promote Heme Acquisition by the Pathogen *

doi: 10.1074/jbc.M116.755934

Figure Lengend Snippet: IsdB heme transfer kinetics, SPR analysis, and cellular uptake of Hb-Hp2-2. A and B, spectral changes over time after mixing of 13 μm sIsdBN1N2 with 1.5 μm metHb (tetramer) (A) or 1.5 μm metHb-Hp1-1 (Hp dimer; two Hb dimers) (B). For comparison, 1.5 μm metHb or 1.5 μm metHb-Hp1-1 mixed 1:1 with PBS are included. By estimating from the absorbance changes, 30.2 ± 0.5% (n = 3) of heme was transferred from metHb to IsdB in 300 s, whereas no measurable transfer to metHb-Hp1-1 was seen. C, relative plateau response of immobilized CD163 binding of Hb-Hp1-1 (○) and (●) Hb-Hp2-2 plotted against the indicated molar ratios of IsdBN1N2. D, sensorgrams of 100 nm Hb-Hp1-1, Hb-Hp2-2, Hb, Hp1-1, or Hp2-2 binding to immobilized IsdBN1N2. E, confocal microscopy images of endocytosis of Hb-Hp by CD163-expressing cells. CHO CD163 cells were incubated with 46 μg/ml Atto-488-labeled Hb-Hp2-2 and IsdBN1N2 in 5-fold molar excess over Hb-Hp. Atto-488-labeled Hb-Hp2-2 is shown in green, DAPI staining of nuclei is shown in blue, and CD163 surface immunostaining (Alexa 647) is shown in red. Mock transfected CHO cells (right panel) were included as a negative control.

Article Snippet: Polyclonal rabbit anti-human CD163 IgG (Dako), earlier shown to specifically inhibit Hb-Hp uptake by CD163 ( 37 ), and irrelevant polyclonal rabbit anti-human IgG (Dako) were included as inhibition controls, both at a concentration of 50 μg/ml.

Techniques: Binding Assay, Confocal Microscopy, Expressing, Incubation, Labeling, Staining, Immunostaining, Transfection, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: The Staphylococcus aureus Protein IsdH Inhibits Host Hemoglobin Scavenging to Promote Heme Acquisition by the Pathogen *

doi: 10.1074/jbc.M116.755934

Figure Lengend Snippet: Model of S. aureus protein IsdH competing for CD163-mediated Hb-Hp uptake. During intravascular hemolysis, the macrophage-specific receptor CD163 normally takes up complexes of Hb-Hp. Uptake and subsequent degradation of heme produces an anti-inflammatory response. The S. aureus protein IsdH binds to Hb-Hp near the binding site of CD163, which hinders the uptake of Hb-Hp. Binding of IsdHN1 to the α-subunit of Hb in the Hb-Hp complex sterically hinders binding of Hb-Hp to CD163. The Hb-Hp-IsdHN1 structure is from Stødkilde et al. (22) (Protein Data Bank code 4WJG), whereas the two SRCR domains from CD163 were modeled from the structure of the SRCR domain of the scavenger receptor A-I (41) (Protein Data Bank code 2OY3). The positively charged residues Arg307 and Lys317 from Hp (blue) interact with negatively charged, calcium-coordinated amino acids in SRCR 2 and 3 in CD163. The calcium atoms are colored red.

Article Snippet: Polyclonal rabbit anti-human CD163 IgG (Dako), earlier shown to specifically inhibit Hb-Hp uptake by CD163 ( 37 ), and irrelevant polyclonal rabbit anti-human IgG (Dako) were included as inhibition controls, both at a concentration of 50 μg/ml.

Techniques: Binding Assay