Materials include the following: Zymosan A (from Saccharomyces cerevisiae), bovine serum albumin (BSA), Roswell Park Memorial Institute Media 1640 (RPMI 1640), PBS (with and without calcium and magnesium), Ficoll-Histopaque 1077-1 and FITC goat anti-mouse IgG (Sigma-Aldrich); fetal bovine serum (Life Technologies). Rat anti-mouse Ly6G-PE and FITC, CD16/CD32 purified (Mouse BD Fc Block) and mouse anti-human CD163-PE (BD Bioscience); F4/80-PE and FITC, PerCP-Cy5.5 CD11b (eBioscience); mouse recombinant granulocyte macrophage colony-simulating factor (GM-CSF), mouse anti-human CD206-APC, HLA-DR-FITC, CD3-FITC and CD19-PE and (Biolegend); 15-LOX-1 (Origene); mouse anti-human ChemR23-PE, human recombinant GM-CSF and hrTNF-α (R&D Systems); human monocyte isolation kits (StemCell Technologies); liquid chromatography (LC)-grade solvents (Fisher Scientific); Eclipse Plus C18 column (Agilent); C18 SPE columns (Waters); EPA (Aldrich); DHA (NuCheck); AT-RvD1 and synthetic and deuterium labeled LM standards (Cayman Chemical).
Pe mouse anti human cd163
Manufactured by BD
Sourced in United States
PE Mouse anti-Human CD163 is a laboratory tool used to detect and analyze the presence of CD163 protein on the surface of human cells. It functions as a specific antibody reagent that binds to the CD163 antigen, allowing for the identification and quantification of cells expressing this target.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Fitc mouse anti human hla dr, by BD (3 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Facscalibur ow cytometer, by BD (3 mentions)
Facsdiva software, by BD (2 mentions)
Apc mouse anti human cd206, by BD (2 mentions)
Apc cy7 mouse anti human cd80, by BD (2 mentions)
Apc mouse anti human hla dr, by BD (2 mentions)
Fitc mouse anti human cd68, by BD (2 mentions)
Anti ccl2, by R&D Systems (2 mentions)
Bv421 mouse anti human cd68, by BD (2 mentions)
Cd86 fitc mouse, by Thermo Fisher Scientific (2 mentions)
F4 80 pe mouse antibodies, by Thermo Fisher Scientific (2 mentions)
Cd206 apc mouse, by Thermo Fisher Scientific (2 mentions)
Bb515 mouse anti human cd86, by BD (2 mentions)
Percoll, by Merck Group (2 mentions)
Software, by FlowJo (2 mentions)
C18 spe column, by Waters Corporation (1 mentions)
Goat anti mouse igg fitc, by Merck Group (1 mentions)
Ficoll histopaque 1077 1, by Merck Group (1 mentions)
13 protocols using pe mouse anti human cd163
1
Isolation and Characterization of Immune Cells
2
Identification of Alveolar Macrophage Subsets
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BAL cells were blocked with 1% BSA containing TruStain fcX (anti-human) antibody (422302; BioLegend), followed by staining with antibodies. Antibodies used: LIVE Dead-eflour506 (65–0866; Invitrogen), Rat anti-human CD11b-APC-Cy7 (101226; BioLegend), Mouse anti-human HLA-DR-PE-Cy7 (560651; BD Biosciences), Mouse anti-human CD206-FITC (551135; BD Biosciences), Mouse anti-human CD169-BB700 (745772; BD Biosciences), and Mouse anti-human CD163-PE (560933; BD Biosciences). Hierarchical gating strategy was used to represent the resident alveolar macrophages as CD11b+HLA-DR++CD206++CD169+CD163++ and monocyte-derived macrophages as CD11b+HLA-DR++CD206++CD169+CD163+. Data was acquired on ARIA (BD Biosciences) using BD FACS DIVA software (version 8.0.1). Data was analyzed using FlowJo (FlowJo LLC) software (Version 10.5.0).
3
Multiparametric Immune Cell Profiling
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Macrophages were processed into single cell suspensions, incubated with antibodies (PE Mouse anti-Human CD163, APC Mouse anti-Human CD206, FITC Mouse anti-Human HLA-DR, APC-Cy7 Mouse anti-Human CD80, all from BD Biosciences, USA) for 1 h at 4 °C. The cells were then washed twice with 4 ml of flow buffer, then centrifuged, and resuspended in 0.5 ml of flow buffer for analysis. Flow cytometry was performed using a FACSCalibur flow cytometer (BD Biosciences, USA). Flow cytometric analysis was performed on FlowJo software (FlowJo, USA).
4
Macrophage and CD8+ T Cell Phenotyping
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Macrophages with or without rISG15 treatment for 12 h were stained with PE mouse anti-human CD163 (BD Bioscience, catalog no. 556018) and FITC mouse anti-human HLA-DR (BD Bioscience, catalog no. 560944). After gating the macrophages, a two-parameter density blot was used to distinguish M2 and M1 cells by creating a plot on CD163 (PE) and HLA-DR (FITC). For detecting the effectors of CD8+ cells, CD8+ cells were stained with PE anti-human CD8a (Biolegend, catalog no. 344705) prior to the intracellular staining with PE/Cyanine 7 anti-human IFN-γ (Biolegend, catalog no. 502527), or FITC anti-human/mouse Granzyme B (Biolegend, catalog no. 515403), or APC anti-human Perforin (Biolegend, catalog no. 308111). The intracellular staining permeabilization wash buffer (Biolegend, catalog no. 421002) is used to permeabilize CD8+ cells and following fixation with fixation buffer (Biolegend, catalog no. 420801). All data were collected using a cytoFLEX flow cytometer (BD Biosciences, USA) and analyzed using FlowJo software (BD Biosciences, USA).
5
Analysis of Macrophage Subsets by Flow Cytometry
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Macrophages analysis was performed using single cell suspensions, following incubation with antibodies (APC Mouse anti-Human CD206, PE Mouse anti-Human CD163, APC-Cy7 Mouse anti-Human CD80, FITC Mouse anti-Human HLA-DR from BD Biosciences, USA) for 1 h at 4 °C. Cells then were washed three times with flow buffer (5 mL), then centrifuged, and resuspended in 1 mL of flow buffer for analysis. Cell analysis was performed using a FACSCalibur flow cytometer (BD Biosciences, USA). Flow cytometric analysis was performed using the FlowJo software.
6
Apoptosis and M2 Macrophage Analysis
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Breast cancer cells were suspended in RPMI‐1640 with 10% FBS at a concentration of 1 × 106 cells/mL. For annexin V‐FITC/propidium iodide (PI) staining, 100 μL cells were transferred to a flow cytometer tube, diluted with PBS, and centrifuged at 300 g for 5 minutes. The supernatant was then removed by decanting and blotting. For each sample, the pellet was suspended in 100 μL of 1× binding buffer, and 5 μL Annexin V‐FITC was added. The samples were incubated in dark for 15 minutes. Before flow cytometer analysis, 400 μL of 1× binding buffer and 5 μL of 50 μg/mL PI were added to each sample. For flow cytometry analysis, PI green fluorescence was dot plotted on the y‐axis and Annexin V‐FITC orange fluorescence on the x‐axis.
Macrophages were washed with PBS. PE mouse anti‐human CD163 (BD Pharmingen) was used to analyze M2 polarization of macrophages. The cells were filtered and resuspended in buffer before sorting. All analyses were conducted on FACSDiva (Version 6.1.3).
Macrophages were washed with PBS. PE mouse anti‐human CD163 (BD Pharmingen) was used to analyze M2 polarization of macrophages. The cells were filtered and resuspended in buffer before sorting. All analyses were conducted on FACSDiva (Version 6.1.3).
7
Characterizing Macrophage Subsets
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Macrophages were harvested from 6-well plates, washed twice with PBS, made into single-cell suspensions, and then incubated with antibodies (FITC Mouse anti-Human CD68, PE Mouse Anti-human CD163, APC Mouse anti-Human HLA-DR, all from BD Biosciences, USA) for 60 min at 4 °C. The stained cells were then washed twice and resuspended in the 200 μl flow buffer for analysis on a FACS Calibur flow cytometer (BD Biosciences, USA) using FlowJo software (FlowJo, USA).
8
Macrophage and Epithelial Cell Sorting
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Detection of target cells by FCM, we used PE Mouse Anti-Human CD163(BD Pharmingen™) to sort the macrophages cells. APC Mouse Anti-Human CD326 staining (BD Pharmingen™) to sort the epithelial cells. Staining was carried out according to the manufacturer’s instructions. FCM was performed on a BD FACS Canto II machine. Data were analyzed using FlowJo™ v.10 software.
9
Macrophage Immunophenotyping by Flow Cytometry
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Macrophages were harvested from 6-well plates, washed twice with PBS, made into single-cell suspensions, and then incubated with antibodies (FITC Mouse anti-Human CD68, PE Mouse Anti-human CD163, APC Mouse anti-Human HLA-DR, all from BD Biosciences, USA) for 60 min at 4°C. The stained cells were then washed twice and resuspended in the 200μl ow buffer for analysis on a FACS Calibur ow cytometer (BD Biosciences, USA) using FlowJo software (FlowJo, USA).
10
Co-culture Macrophage Phenotyping Protocol
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THP-1 macrophages were co-cultured with stably transfected CRC cells along with or without neutralizing antibodies to CCL2 (anti-CCL2; R&D Systems). Then these macrophages were processed into single cell suspensions, incubated with antibodies (BV421 Mouse anti-Human CD68, BB515 Mouse anti-Human CD86, PE Mouse anti-Human CD163, all from BD (BD Biosciences, San Jose, CA, USA) for 1 h at 4 ℃.
Mouse macrophages were then stained with CD206-APC (mouse), CD86-FITC (mouse), F4/80-PE (mouse) antibodies (eBiosciences, San Diego, CA, USA), respectively. Macrophages of nude mice subcutaneous tumor were separated and obtained using Percoll (Sigma-Aldrich, St. Louis, MO, USA) following the instruction. Flow cytometry was performed using a FACS Calibur ow cytometer (BD Biosciences). Flow cytometric analysis was performed on FlowJo software (FlowJo, Ashland, OR, USA).
Mouse macrophages were then stained with CD206-APC (mouse), CD86-FITC (mouse), F4/80-PE (mouse) antibodies (eBiosciences, San Diego, CA, USA), respectively. Macrophages of nude mice subcutaneous tumor were separated and obtained using Percoll (Sigma-Aldrich, St. Louis, MO, USA) following the instruction. Flow cytometry was performed using a FACS Calibur ow cytometer (BD Biosciences). Flow cytometric analysis was performed on FlowJo software (FlowJo, Ashland, OR, USA).
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