25-1639-41

antibody from Invitrogen Antibodies

Targeting: CD163 M130, MM130, SCARI1

Provider product page for 25-1639-41

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Antibody data

Antibody Data
Antigen structure
References [17]
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Validations
Flow cytometry [1]
Other assay [20]

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Product number: 25-1639-41 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD163 Monoclonal Antibody (eBioGHI/61 (GHI/61)), PE-Cyanine7, eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The eBioGHI/61 monoclonal antibody reacts with human CD163 (Haemoglobin Scavenger Receptor, M130), a member of the macrophage scavenger receptor family. CD163 is expressed by tissue macrophages, circulating blood monocytes, and some reports suggest that it may also be expressed by haematopoietic progenitor cells. CD163 functions as a high affinity scavenger receptor for the complex of haemoglobin and haptoglobin which forms to clear haemoglobin from the blood. The high affinity for CD163 is specific for the haemoglobin-haptoglobin complex, whereas haemoglobin or haptoglobin display no to low affinity toward CD163. Upon ligand binding, CD163 initiates signal transduction which leads to the production of IL-6 and IL-10. Furthermore, it has been demonstrated that cytokines such as IL-6 and IL-10 can induce expression of CD163, whereas proinflammatory stimuli, such as LPS or TNF alpha, can cause shedding of a soluble form of CD163, whose function is unclear.
Antibody clone number: eBioGHI/61 (GHI/61)
Concentration: 5 µL/Test

CD163 protein structure - 25-1639-41 shown in red.

Supportive validation

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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 5 Interleukin-25 (IL-25) does not alter the expression of CD163 and CD86 in human CD14 + cells. (a) Representative dot-plots showing the expression of CD163 and CD86 in CD14 + cells pre-incubated with medium, IL-25 (50 ng/mL) or IL-10 (25 ng/ml) for 30 min and then either left unstimulated (Uns) or stimulated with lipopolysaccharide (LPS; 100 ng/ml) or tumour necrosis factor-alpha (TNF-alpha; 10 ng/ml). After 12 hr, CD23 and CD163 expression was assessed by flow cytometry. Numbers indicate the percentage of positive cells within the designated quadrants. One of three representative experiments in which similar results were obtained is shown. (b) Induction of CD163 by IL-10 is not influenced by IL-25. Representative histograms showing the percentage of CD14 + cells positive for CD163. Cells were pre-incubated with IL-25 (50 ng/ml) for 6 hr and then stimulated with IL-10 (25 ng/ml) for a further 24 hr. Data indicate the mean +- SD of three experiments.

Supportive validation

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Experimental details: Fig. 6 Surface expression of key markers of microglia. a Flow cytometry analysis was used to measure the surface expression of CD11b, CD14, CD68, CD16, CD32, CD64, CD163, P2RY12, and TGFbetaR on the representative immortalized cell line hmuglia 1A1. In each experiment, 100,000 cells were resuspended in 1 mL of cold PBS in the presence of 0.5 mug of the antibody or isotype control for 20 min on ice. Appropriate secondary antibodies were used in the absence of fluorophore-conjugated primary antibody, and the cell-antibody complexes were centrifuged and resuspended in PBS. In each FACS profile, the gray distributions represent the proportion of cells bound to the isotype control, whereas the red distributions represent the proportions of cells bound by the target antibody. b Quantification of the abovementioned markers as well as GFAP, CD4, and CCR5 on the surface of primary human astrocytes and immortalized microglia, as indicated

Supportive validation

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Experimental details: Figure 3 CD64 and CD163 expression on monocytes, granulocytes, and lymphocytes.

Supportive validation

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Experimental details: Figure 2 miR-720 suppresses the expression of M2 phenotypes ( A ) Real-time PCR analysis of miR-720 expression in control vector or pLV- miR-720 -infected THP-1 cells. ( B ) Control or pLV- miR-720 -infected THP-1 cells were induced macrophage differentiation by PMA (M0) and then treated with IFN-gamma plus LPS (M1) or IL-4 plus IL-13 (M2) to induce polarization. The macrophage marker CD68, M1 marker CD86 and M2 marker CD163 were analysed by flow cytometry respectively. ( C ) Detection of M1 cytokine TNF-alpha and IL-6 or ( D ) M2 cytokine IL-10 and chemokine CCL17 by real-time PCR in THP-1 cells used in ( B ). Values are means +- S.D. ** P

Supportive validation

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Experimental details: Figure 3 miR-720 targets GATA3 to regulate M2 polarization ( A ) The 3'UTR of the GATA mRNA containing the putative miR-720 -binding sequence is shown. Mutation was generated in the GATA 3'UTR sequence at the complementary site for the seed region of miR-720 . ( B ) HEK-293 cells were co-transfected with wild-type or mutant GATA3 3'UTR luciferase reporter together with control or pLV- miR-720 construct. Luciferase assays were performed after 24 h. ( C ) The mRNA or ( D ) protein expression levels of GATA3 in both control and pLV- miR-720 -infected THP-1 cells were analysed by real-time PCR or western blotting respectively. ( E ) Western blotting analysis of GATA3 expression in control, pLV- miR-720 -infected or pLV- miR-720 -infected plus FLAG-GATA3 transfected THP-1 cells. ( F ) The cells used in ( E ) were polarized with IL-4/IL-13 and the M2 marker CD163 was analysed by flow cytometry. ( F ) The expression of IL-10, CCL17 and Arginase-1 in the cells used in ( E ) was analysed by real-time PCR. Values are means +- S.D. ** P

Supportive validation

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Experimental details: EV2 Flow cytometry analysis of macrophage populations A Representative images showing FACS dot plots for HLA-DR + /CD86 + macrophages. Average frequencies of 4 donors are shown. B Quantification of HLA-DR + /CD86 + macrophages across macrophage populations. Lines and error bars represent mean +- SD from two independent experiments (2 macrophage donors each). C, D Expression levels (geometric mean of fluorescent intensity, gMFI) of (C) HLA-DR and (D) CD86 in HLA-DR + /CD86 + macrophages across macrophage populations. Lines and error bars represent mean +- SD from two independent experiments (2 macrophage donors each). E Representative images showing FACS dot plots for CD206 + /CD163 + macrophages. Average frequencies of 4 donors are shown. F Quantification of CD206 + /CD163 + macrophages across macrophage populations. Lines and error bars represent mean +- SD from two independent experiments (2 macrophage donors each). G, H Expression levels (geometric mean of fluorescent intensity, gMFI) of (G) CD206 and (H) CD163 in CD206 + /CD163 + macrophages across macrophage populations. Lines and error bars represent mean +- SD from two independent experiments (2 macrophage donors each). Data information: Macrophage donors are indicated as D22-D25. M2D-M2-differentiated, M1A-M1-activated, M2D-M2-differentiated, M2A-M2-activated. * P < 0.05 as measured by one-way ANOVA with uncorrected Fisher''s LSD post hoc test (exact P values are shown in Table EV3). Source data are available online f

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 5 Interleukin-25 (IL-25) does not alter the expression of CD163 and CD86 in human CD14 + cells. (a) Representative dot-plots showing the expression of CD163 and CD86 in CD14 + cells pre-incubated with medium, IL-25 (50 ng/mL) or IL-10 (25 ng/ml) for 30 min and then either left unstimulated (Uns) or stimulated with lipopolysaccharide (LPS; 100 ng/ml) or tumour necrosis factor-alpha (TNF-alpha; 10 ng/ml). After 12 hr, CD23 and CD163 expression was assessed by flow cytometry. Numbers indicate the percentage of positive cells within the designated quadrants. One of three representative experiments in which similar results were obtained is shown. (b) Induction of CD163 by IL-10 is not influenced by IL-25. Representative histograms showing the percentage of CD14 + cells positive for CD163. Cells were pre-incubated with IL-25 (50 ng/ml) for 6 hr and then stimulated with IL-10 (25 ng/ml) for a further 24 hr. Data indicate the mean +- SD of three experiments.

Supportive validation

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Experimental details: Fig. 5 Expression of AAT in human MPhi. A Representative AAT western blot analysis of human MPhi supernatant and MPhi lysates. Human serum was used as a positive control. Vinculin band in cellular lysates served as loading control. B ELISA quantification of AAT secretion by human MPhi. n = 3. C Representative May-Grunwald-Giemsa staining of MPhi cytospins. Scale bar = 20 mum. D Representative flow cytometric analysis of myeloid and MPhi-specific surface marker expression by human MPhi. E GM-CSF uptake from cell culture medium by MPhi. A well without cells was used as a negative control. n = 3. All data represent independent cell isolations and transductions.

Supportive validation

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Experimental details: The correlation between TAMs and HNSCC prognosis and the establishment of HOTTIP overexpression and knockdown exosomes in M1. A The infiltration levels of immune cells in HNSCC. B Prediction of overall survival according to high- and low-infiltration levels of M0, M1 and M2 macrophages. THP-1-derived M0, M1 and M2 macrophages were confirmed by flow cytometry using ( C ) CD14 plus CD86 and ( D ) CD14 plus CD163 ( n = 3). E The exosomes were extracted from three types of macrophages and confirmed by electronic microscopy and Western blot analysis using CD9 and CD63. F RT-qPCR was conducted to test the expression of HOTTIP in M0, M1 and M2 macrophages ( n = 3). RT-qPCR was conducted to test the expression of HOTTIP in wild type M1 macrophages, HOTTIP-overexpressed and HOTTIP-knockdown M1 macrophages ( G ) and their exosomes ( H ) ( n = 3). Data are presented as mean +- SD. Results were analyzed by One-way ANOVA with a post hoc t -test. Significance: * P < 0.05, ** P < 0.01, *** P < 0.001.

Supportive validation

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Experimental details: Cancer cells expressing HOTTIP and M1 exosomes reprogram the circulating monocytes into M1 phenotype. A-D Flow cytometry assay detected CD14 + CD86 + M1 and CD14 + CD163 + M2 phenotype in circulating blood of nude mice bearing with HOTTIP-knockdown Hep-2 cells ( n = 3). HOTTIP-knockdown Hep-2 cells suppressed the polarization of M1 monocytes ( A , C ) but induced the polarization of M2 ( B , D ). E - H Flow cytometry assay detected M1 and M2 phenotypes in the blood of tumor-bearing nude mice treated by M1 exosomes ( n = 3). M1 exosomes induced the polarization of M1 monocytes ( E , G ) but inhibited the polarization of M2 ( F , H ). Data are presented as mean +- SD. Results were analyzed by One-way ANOVA with a post hoc test. Significance: ns not significant, ** P < 0.01, *** P < 0.001.

Supportive validation

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Experimental details: Fig. 4 RAD18-driven YAP activation in TNBC cells promotes M2-like TAM polarization. A The Timer software showed that high-expressed RAD18 was correlated with high-level macrophage infiltration in BRCA. RAD18 expression level was proportional to M2-like TAM infiltration, whereas inversely proportional to M1-like TAM infiltration. B Schematic model of the co-culture system of macrophages and TNBC cells; THP-1 cells were stimulated by PMA for 48 h to form M0 macrophages, then MDA-MB-231 cells stably expressing shNC or shRAD18 were co-cultured with M0 macrophages for 72 h. The M0 alone were used as a negative control. C Detection of the expression of CD86 (M1-like macrophage marker) and CD163 (M2-like macrophage marker) in co-cultured macrophages by Flow cytometry analysis. D WB detection of protein expression of CD86 and CD163 in macrophages after co-culture. E The expressions of CD86 (red) and CD163 (green) in macrophages were detected using immunofluorescence staining (400x). Cell nuclei were stained with DAPI (blue). F The expression of CD86 and CD163 in macrophages co-cultured with the shNC TNBC treated with verteporfin or the shRAD18 TNBC treated with YAP OE plasmid were detected by flow cytometry analysis. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were performed independently at least three times.

Supportive validation

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Experimental details: Fig. 1 M2-TAMs maintain the properties of GSCs. Kaplan-Meier survival analysis of progression-free survival ( A ) and overall survival ( B ) of GBM patients with low or high Iba1 expression using GEPIA ( http://gepia2.cancer-pku.cn/#index ) in TCGA database ( https://portal.gdc.cancer.gov/ ). Kaplan-Meier survival analysis of progression-free survival ( C ) and overall survival ( D ) of GBM patients with high or low CD163 expression using GEPIA in TCGA database. E RT-qPCR was utilized to analyze the expression of CD163 in the CD11b + /CD163 + and CD11b + /CD163 - TAMs from 6 GBM patients sorted by FACS. F The morphology of GSCs. G Western blot analysis of stem cell-related proteins CD133, Nestin, Oct4, Sox2, and GFAP. H Representative images showing neurosphere formation of GSCs along with the statistics of GSC neurosphere formation rate and diameter. I Expression of stem cell-related protein CD133, Nestin, Oct4, Sox2 and GFAP in GSCs measured by Western blot analysis. * p < 0.05 vs. CD163, GBMs or control TAMs. Measurement data were depicted as mean +- standard deviation, and comparison of data between two groups was conducted by unpaired t test. Cell experiments were repeated three times independently.

Supportive validation

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Experimental details: Fig. 2 M2-TAM-derived exosomes enhance the stemness of GSCs. A TEM images of M2-TAM-derived exosomes. The presence of CD63, CD206, and CD163 in M2-TAM-derived exosomes analyzed by flow cytometric ( B ) and Western blot ( C ) analyses. D Fluorescence images of GSCs co-cultured with Dil-labeled M2-TAM-derived exosomes with nuclei stained with DAPI (blue). E Representative images showing neurosphere formation of GSCs along with the statistics of GSC formation rate and diameter of spheres. F Expression of stem cell-related protein CD133, Nestin, Oct4, Sox2, and GFAP in GSCs measured by Western blot analysis. G HE staining images of xenograft tumors from mice. H The representative immunohistochemical images of Sox2 in xenograft tumors and the percentage of GSCs labeled by Sox2. I Kaplan-Meier survive curve of tumor-bearing mice. n = 10. * p < 0.05 vs. the control EXO group. Measurement data were depicted as mean +- standard deviation, comparison of that between two groups was conducted by unpaired t test. Cell experiments were repeated three times independently.

Supportive validation

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Experimental details: Figure 3 CFT073 wt suppresses M1 but promotes M2 macrophage polarization in vitro. ( A , B ) The expression of M1 surface markers CD80 and CD86 in THP-1 and J774A.1 was detected by flow cytometry. ( C , D ) The expression of M2 surface markers CD163 and CD206 in THP-1 and J774A.1 was analyzed by flow cytometry. ( E ) The production of NO in THP-1 and J774A.1 was measured by Griess method. ( F , G ) The iNOS protein levels and bands' gray-scale analyses in THP-1 and J774A.1, respectively. ( H ) The protein levels of Arg-1 in THP-1 and J774A.1 were detected by ELISA. ( I , J ) The protein levels of Arg-1 were detected by Western blot and bands' gray-scale analyses in THP-1 and J774A.1, respectively. Gray scale in the control group was set as 1.0. Mean +- SD of three independent experiments are shown. *: p < 0.05; **: p < 0.01. NS: not significant.

Supportive validation

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Experimental details: Figure 4 rTcpC inhibits M1 markers CD80 and CD86 but promotes the expression of M2 markers CD163 and CD206 in macrophages. ( A - D ) The effects of rTcpC on M1 surface markers CD80 and CD86 detected by flow cytometry and FI were analyzed by FlowJo v10.0.7 software. ( E - H ) The surface markers CD163 and CD206 of M2 polarized macrophages were detected by flow cytometry. Mean +- SD of three independent experiments are shown. **: p < 0.01.

Supportive validation

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Experimental details: Figure 1 Orthotopic liver transplantation increases KC activation and TIM-4 expression. (A) KCs were isolated from mice in the sham and LT groups 24, 48 and 72 h following surgery. Cells were double stained with PE-CD14 and APC-CD163 and quantified using flow cytometry. (B) Liver samples were obtained from mice in the sham and LT groups and assessed using immunohistochemistry (magnification, x400). Activated hepatic KCs are stained brown. (C) KCs were isolated from mice in the sham and LT groups 1, 3 and 7 days following surgery and TIM-4 expression was analyzed using western blotting. Data are presented as the mean +- standard deviation. a P

Supportive validation

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Experimental details: Figure 7 BCA regulate MPhi polarization in vitro and in vivo . (A,B) Raw246.7 cells were treated with 4 mug/ml BCA for 2 h. Then, the cells were infected with Salmonella at MOI = 10:1 for 2 h. (A) The expression of surface markers (CCR7, CD86, CD163, and CD206) was determined by flow cytometry. The results are presented as MFI. (B) TNF-alpha and IL-10 in the supernatants were detected by ELISA. ** P < 0.01, *** P < 0.001. The data are representative of three experiments with similar results. (C,D) Mice were infected by intragastric administration of an overnight culture of Salmonella (10 5 bacteria in 0.1 ml PBS) through a gavage tube and then treated with 6.25 mg/kg BCA intragastrically by gavage daily (5 mice for each group). On the 5th day p.i., the mice were sacrificed and the peritoneal fluid was collected. (C) The expression of surface markers (CCR7, CD86, CD163, and CD206) in mouse peritoneal MPhis were determined by flow cytometry The results are presented as MFI. (D) TNF-alpha and IL-10 in ascitic fluid were detected by ELISA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Supportive validation

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Experimental details: Figure 3 Generation of patient specific iPSC-derived macrophages. Patient iPSCs have been differentiated into macrophages and compared to macrophages from a healthy iPSC line (hCD34_iPSC16). ( A ) Microscopic analysis of patient iPSCs in cytospin images after Pappenheim staining (left, scale bar = 20 um) or in brightfield images (middle scale bar 200 um, right scale bar = 100 um). ( B ) Representative flow cytometric analysis of CD11b, CD14, CD163 and CD45 expression on patient iPSCs and healthy macrophages of two independent experiments. Blue: Isotype. Pink: Surface marker. FC = fold change of the median fluorescent intensity. ( C ) Flow cytometric analysis of IFN-gammaR1 (top) and IFN-gammaR2 (bottom) expression on healthy and patient iPSC-derived macrophages. Blue: Isotype. Pink: Surface marker. Expression has been quantified by plotting the difference of the median fluorescent intensity (DeltaMFI). Each dot represents macrophages from an independent harvest and from at least three independent differentiations ( n = 4-7, mean +- SD, Kruskal-Wallis with Dunn''s multiple comparison). Red line shows DeltaMFI of 0. ( D ) GM-CSF clearance of healthy and patient iPSC-derived macrophages over a time of 48 h. Concentrations have been normalized to control well containing no cells (media only) ( n = 3, mean +- SD; each dot represents macrophages from an independent harvest and from at least three independent differentiations). ( E ) Representative flow cytometric (top) and microsco