Anti cd163 pe

Manufactured by BioLegend

Sourced in Germany

Anti-CD163-PE is a fluorochrome-conjugated antibody that recognizes the CD163 antigen. CD163 is a scavenger receptor expressed on the surface of monocytes and macrophages. The PE fluorochrome allows for the detection and analysis of CD163-positive cells using flow cytometry or other fluorescence-based techniques.

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Close spelling variants of this product

PE-Cy7 anti-CD163 PE-Cy7 anti-human CD163 PE anti-human CD163 PE anti-mouse CD163 PE Mouse anti-Human CD163 PE-conjugated anti-human CD163 antibody PE-conjugated anti-human CD163 CD163-PE Anti-CD163 CD163

10 protocols using anti cd163 pe

Sarcoma Tumor Dissociation and Immunophenotyping

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Sarcomas were grown in mice until approximately 1 × 1 × 1 cm in size. Tumors were resected and enzymatically digested. Where applicable, tumor cells were FACS-sorted based on the expression of the fluorescent marker dsRED.
Human sarcoma samples were minced and then enzymatically and mechanically digested using the 37C_h_TDK_2 protocol on the Miltenyi gentleMACS with Tumor Dissociation Kit for Human cells (Miltenyi Biotec, Auburn, CA). Cell suspensions were washed in DPBS with 0.04% BSA and filtered through 70 μm strainers (Bioland Scientific LLC, Paramount, CA). Red blood cells were lysed with ACK buffer prior to staining with the following surface antibodies: anti-CD45 Pacific Blue, anti-CD11b APC, anti-CD163 PE, anti-CD3 FITC, anti-CD4 PE, anti-CD8 APC, anti-CD56 APC Cy7, anti-CD19 FITC (all purchased from Biolegend).

Tessaro F.H., Ko E.Y., De Simone M., Piras R., Broz M.T., Goodridge H.S., Balzer B., Shiao S.L, & Guarnerio J. (2022). Single-cell RNA-seq of a soft-tissue sarcoma model reveals the critical role of tumor-expressed MIF in shaping macrophage heterogeneity. Cell reports, 39(12), 110977.

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Characterization of DRG Myeloid Cells

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DRG single-cell suspensions were blocked for non-specific antibody-binding with anti-CD16/32 Fc-Block (BioLegend 101320, 1:100) and incubated in an antibody cocktail consisting of anti-CD163-PE (BioLegend 156703, 1:200), anti-TLR4-PE/Cy7 (BioLegend 117609, 1:200), anti-CD206-PerCP/Cy5.5 (BioLegend 141715, 1:200), anti-CD11b-APCCy7 (BioLegend 101225, 1:200), anti-CD68-Alexa700 (BioLegend 137025, 1:200), anti-CD80-Bv421 (BioLegend 104725, 1:200), anti-CD86-Bv510 (BioLegend 105039, 1:200), anti-Gr1-Bv605 (BioLegend 10844, 1:200), anti-F4/80-Bv785 (BioLegend 123141, 1:200), and anti-CX3CR1-FITC (BioLegend 149019, 1:200) for 20 min at 4 °C. Exclusion of dead cells was achieved by resuspending cells in FACS buffer containing 10 nM TO-PRO-3 (Invitrogen, Carlsbad, CA, USA, T3605). Flow cytometry from the samples was performed using a BD LSRFortessa device and BD FACSDiva Software (BD Biosciences). Raw data were analyzed using the CytoExplorer R package version 1.1.0 [46 ].

Choconta J.L., Labi V., Dumbraveanu C., Kalpachidou T., Kummer K.K, & Kress M. (2023). Age-related neuroimmune signatures in dorsal root ganglia of a Fabry disease mouse model. Immunity & Ageing : I & A, 20, 22.

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Flow Cytometric Analysis of Macrophage Surface Markers

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Macrophages were harvested, washed with PBS, then pelleted at 300 × g at 4°C for 5 min. Cells were blocked with 2% Fc Receptor Binding Inhibitor (eBioscience, Frankfurt, Germany) in PBS for 10 min on ice. Afterward, the following antibody mix was added in 100 ml PBS: anti-CD80-APC, anti-CD86-FITC (BD Biosciences, Heidelberg, Germany), anti-CD163-PE, and anti-CD206-PE-Cy5 (BioLegend, San Diego, CA, USA), followed by incubation for 20 min on ice in the dark. Samples were washed and analyzed by flow cytometry using an LSRII Fortessa cell analyzer (BD Biosciences).

Snodgrass R.G., Zezina E., Namgaladze D., Gupta S., Angioni C., Geisslinger G., Lütjohann D, & Brüne B. (2018). A Novel Function for 15-Lipoxygenases in Cholesterol Homeostasis and CCL17 Production in Human Macrophages. Frontiers in Immunology, 9, 1906.

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Macrophage Phenotype Analysis by Flow Cytometry

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At day 2, 4 and 8 the differentiated macrophages were carefully scraped from the CFs or well ground of the culture plates and seeded (5 × 10⁵ cells per well) into 96-well plates. Cells were washed with PBS. Subsequently the Fc receptors were blocked by incubation in a buffer containing 10 µg/ml heat-aggravated human immunoglobulin G (IgG) (Sigma, Deisenhofen, Germany). For extracellular staining the following antibodies were used: anti-CD163-PE (BioLegend, San Diego, CA, USA), anti-CD206-PE, (BD Pharmingen, Heidelberg, Germany) and anti-CCR7-FITC (R&D Systems) and the respective isotype controls to define the unspecific background staining. For intracellular staining cells were fixed, permeabilized using the BD Cytofix/Cytoperm fixation/permeabilization kit (BD Bioscience, Heidelberg, Germany) and anti-CD68-APC (BioLegend) or the respective isotype control was applied. Stained cells were washed three times and resuspended in PBS. Sample acquisition was performed by flow cytometry (FACS Calibur, Becton Dickinson, Heidelberg, Germany) and geometric mean fluorescence intensities (MFI) were calculated by CellQuest Pro software (Becton Dickinson).

Stenberg L., Stößel M., Ronchi G., Geuna S., Yin Y., Mommert S., Mårtensson L., Metzen J., Grothe C., Dahlin L.B, & Haastert-Talini K. (2017). Regeneration of long-distance peripheral nerve defects after delayed reconstruction in healthy and diabetic rats is supported by immunomodulatory chitosan nerve guides. BMC Neuroscience, 18, 53.

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Quantifying M2 Macrophage Markers

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Cells were resuspended in 50 μl of staining buffer (PBS:FBS = 1000:1) with 2 μl of Fc block (422302; Biolegend, USA) for 15 min at 4°C. Then, 1.5 μl of anti‐CD163‐PE (333605; Biolegend) and 1.5 μl of anti‐CD206‐APC (321109; Biolegend) were added to the reaction for 30 min at 4°C. After 30 min, THP‐1 were washed twice with staining buffer, and 1% formaldehyde was used for fixation at 4°C. The data were detected by flow cytometry (Beckman Coulter).

Liu Y., Wang X., Zhu Y., Cao Y., Wang L., Li F., Zhang Y., Li Y., Zhang Z., Luo J., Deng X., Peng C., Wei G., Chen H, & Shen B. (2022). The CTCF/LncRNA‐PACERR complex recruits E1A binding protein p300 to induce pro‐tumour macrophages in pancreatic ductal adenocarcinoma via directly regulating PTGS2 expression. Clinical and Translational Medicine, 12(2), e654.

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Sarcoma Tumor Dissociation and Immunophenotyping

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Identification of CD11b+CD163+ Macrophages

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To detect CD11b⁺CD163⁺macrophages, anti-CD11b-FITC (Biolegend, 301330) and anti-CD163-PE (Biolegend, 333606) monoclonal antibodies were applied to sort cells with Cytoflex flow cytometer (Beckman Coulter, Krefeld, Germany). Cells were gated based on FSC and SSC characteristics. All data was analyzed with FlowJo software (FlowJo 10.8.1; FlowJo, Ashland, OR, USA).

Huang S., Zhang P., Yin N., Xu Z., Liu X., Wu A., Zhang X., Li Z., Zhang Z., Zhong T., Liu L., Shi Y, & Dong J. (2024). Glioblastoma stem cell-derived exosomal miR-374b-3p promotes tumor angiogenesis and progression through inducing M2 macrophages polarization. iScience, 27(3), 109270.

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Comprehensive Flow Cytometry Immunophenotyping

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For flow cytometry analysis, tumor and blood samples were stained with a cocktail of monoclonal mouse anti-human conjugated antibodies (mAbs): anti-CD45-PercP (clone HI30), anti-CD3-PercP (HIT3a), anti-CD3-APC (UCHT1), anti-CD19-PE (HIB19), anti-CD15-PE (HI98), anti-CD161-FITC (HP-3G10), anti-CD4-FITC (OKT4), anti-CD8-PE (HIT8a), anti-HLA-DR-APC (L243), anti-CD127-PE-Cy7 (A019D5), anti-CD1c-APC-Cy7 (L161), anti-CD163-PE (GHI/61), anti-CD206-APC-Cy7 (15–2), anti-PD-1-FITC (EH12.2H7), anti-PD-L1-APC (29E2A3), anti-CTLA4-PE (L3D10), anti-CD69-APC-Cy7 (FN50), anti-Tim3-APC (F38-2E2), anti-IL-8-APC (E8N1), anti-IFN-γ-PE (4S.B3), anti-IFN-γ-APC-Cy7 (4S.B3), anti-Granzyme B-FITC (QA16A02), anti-IL-1β-FITC (JK1B-1), anti-IL-2-PE-Cy7 (MQ1-17H12), anti-IL-6-APC (MQ2-13A5), anti-IL-17-FITC (BL168), anti-IL-23/IL-12-PE (C11.5), anti-TGF-β-APC (TW4-6H10), all from Biolegend; anti-IDO-PE (eyedio) and anti-IL-10-FITC (BT-10), both from eBioscience; anti-CD25-PE (MEM-181) and anti-CD11b-FITC (LT11) from ImmunoTools.
The antibodies used for immunofluorescence were: mouse monoclonal anti-human CD8 (32-M4) from Santa Cruz Biotechnology and rabbit polyclonal anti-human HLA-DRA from Sigma Aldrich.

Saraiva D.P., Jacinto A., Borralho P., Braga S, & Cabral M.G. (2018). HLA-DR in Cytotoxic T Lymphocytes Predicts Breast Cancer Patients' Response to Neoadjuvant Chemotherapy. Frontiers in Immunology, 9, 2605.

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Intracellular IFN-α expression analysis

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Intracellular IFN-α staining was performed according to the intracellular staining protocol from BD Bioscience using anti-human IFN-α antibody APC (Miltenyi Biotec). Surface marker staining with anti-CD14 V450 (BD Bioscience), anti-CD163 PE, anti-CD206 PE-Cy7, and anti-CD209 APC (BioLegend) was performed for 20 min at 4°C. Data were acquired on a LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star).

Paijo J., Döring M., Spanier J., Grabski E., Nooruzzaman M., Schmidt T., Witte G., Messerle M., Hornung V., Kaever V, & Kalinke U. (2016). cGAS Senses Human Cytomegalovirus and Induces Type I Interferon Responses in Human Monocyte-Derived Cells. PLoS Pathogens, 12(4), e1005546.

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Macrophage Polarization by Cytokines and RABV

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Mature macrophages (n = 6 donors) were stimulated for 48 hours with complete medium containing IFN-g (20 ng/mL, R&D Systems) and LPS (100 ng/mL, Sigma Aldrich), with IL-4 (20 ng/mL, R&D Systems), or with IL-1 (20 ng/mL R&D Systems), to induce the M1, M2a or M2c phenotype, respectively. To investigate the effect of rabies virus on macrophage polarization, macrophages were stimulated with complete medium containing RABV (MOI of 10). Expression of macrophage phenotypical markers was investigated by flow cytometry. Non-polarized macrophages, cultured for 48 hours with complete medium without additional cytokines, were taken along as controls for each donor.
Macrophages were dissociated from the wells using Accutase (Merck Millipore) and were washed twice with PBS before staining for 30 minutes with the fixable viability dye ZombieViolet (Biolegend). Cells were fixed with 4 % PFA for 15 minutes, and after Fc receptor blocking with Human TruStain FcX (Biolegend), cells were stained with the following antibodies in FACS buffer (PBS with 2 % fetal calf serum, 0.2 mM EDTA, 0.01% sodium azide): anti-CD80-FITC, anti-HLA-DR-APC-Cy7, anti-PD-L1-APC, anti-CD163-PE, anti-CD200R-PE-Cy7 and anti-CD206 (BV786) (All Biolegend). Mean fluorescent intensities were quantified by flow cytometry using a BD Lyric flow cytometer (BD Biosciences) and the data were analyzed using FlowJo V10.6.2.

Embregts C.W.E., Begeman L., Voesenek C.J., Martina B., Koopmans M., Kuiken T., & GeurtsvanKessel C.H. (2021). Street RABV induces the cholinergic anti-inflammatory pathway in human monocyte-derived macrophages by binding to nAChr ɑ7, and upregulates the M2-c marker CD163.

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