Peripheral blood mononuclear cells (PBMCs) were isolated from patients by Ficoll-Paque PLUS (GE Healthcare, Munich, Germany). Single-cells suspensions were prepared as previously described45 (link). Cells were washed twice with flow cytometry wash buffer. The fluorochrome-coupled antibodies were applied: anti-CD14, anti-CD11c and anti-CD163 (both from BD Biosciences, Heidelberg, Germany). Samples were assessed by flow cytometry using a BD FACS Calibur device and analyzed by FlowJo software.
Anti cd163
Manufactured by BD
Sourced in United States
Anti-CD163 is a laboratory reagent used for the detection and analysis of the CD163 protein, which is expressed on the surface of certain immune cells. It can be used in various research and diagnostic applications that involve the study of CD163-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria 3, by BD (2 mentions)
Anti cd45, by BD (2 mentions)
Anti cd11b, by BD (2 mentions)
Anti cd11c, by BD (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Laser confocal microscope, by Leica (1 mentions)
Anti cd86, by BD (1 mentions)
Dylight 488 goat anti rabbit igg, by Abbkine (1 mentions)
Human serum, by Merck Group (1 mentions)
Anti cd66b, by BD (1 mentions)
Zombie aqua fixable viability kit, by BioLegend (1 mentions)
Hanks balanced salt solution (hbss), by Euroclone (1 mentions)
Syto 16 green fluorescent nucleic acid stain, by Thermo Fisher Scientific (1 mentions)
Anti cd66b, by BioLegend (1 mentions)
Collagenase type 4, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Hepes, by Lonza (1 mentions)
Ack lysing buffer, by Lonza (1 mentions)
Anti cd68, by BD (1 mentions)
6 protocols using anti cd163
1
PBMC Isolation and Characterization
2
Decidual Macrophage Responses to Toxoplasma Infection
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Purified, human decidual macrophages from uninfected, infected, and LILRB4-neutralized infected groups were cultured for 24 h after T. gondii infection and then fixed in 4% paraformaldehyde for 15 min and blocked with goat serum for 1 h. Cells were then incubated overnight at 4°C with anti-LILRB4 (Santa Cruz, Germany) and anti-CD163 (BD, USA), or anti-LILRB4 and anti-CD86 (BD, USA) antibodies. DyLight 488-goat anti-rabbit IgG (Abbkine, USA) was used as a secondary antibody for anti-LILRB4 antibody, and DyLight 649-goat anti-mouse IgG (Abbkine, USA) was used as a secondary antibody for anti-CD163 and anti-CD86. Cells were incubated with appropriate concentrations of secondary antibodies at 37°C for 1 h and were subsequently stained with the nucleic acid stain 4′,6-diamidino-2-phenylindole for 15 min. Finally, cells were observed using a laser confocal microscope (Leica, Germany).
3
Single-cell sorting for MP and CD45+ analysis
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Single-cell suspensions were then preincubated with a blocking solution containing 1% human serum (Sigma-Aldrich, catalog no. H3667) in saline solution and stained with fluorophore-conjugated primary antibodies at room temperature in the dark for 15 minutes: anti-CD45 (BD Biosciences, clone HI30, RRID:AB_1645452), anti-CD66b (BD Biosciences, clone G10F5, RRID:AB_396067), and anti-CD163 (BD Biosciences, clone GHI/61, RRID:AB_2737697). Cell viability was assessed using Zombie Aqua Fixable Viability Kit (BioLegend, catalog no. 423101). MP and CD45+ cells were sorted on a FACSAria III (BD Biosciences) as shown in Supplementary Fig. S1B.
4
Isolation of Macrophage Subsets from Colorectal Liver Metastases
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Macrophages were FACS sorted from surgically resected CLM tissues from five patients. Single-cell suspensions were obtained by manually mincing tissue into small fragments and incubating the fragmented tissues for 1 h at 37°C in HBSS (Euroclone) containing 1 mg/ml Type IV Collagenase (Sigma-Aldrich), 2% FBS (Sigma-Aldrich), 50 μg/ml DNase I (Sigma-Aldrich), and 10 mM Hepes (Lonza). The resulting cell suspension was filtered through a 70-μm cell strainer and erythrocytes were lysed with ACK Lysing Buffer (Lonza). Cells were then incubated with a blocking solution containing 1% human serum in saline solution and stained with the following fluorophore-conjugated primary antibodies: anti-CD45 (BD Biosciences; clone HI30), anti-CD11b (BD Biosciences; clone ICRF44), anti-CD16 (BioLegend; clone 3G8), anti-CD14 (BD Biosciences; clone M5E2), anti-CD66b (BioLegend; clone G10F5), and anti-CD163 (BD Biosciences; clone GHI/61). Fixable Viability Stain 700 fluorescent dye (BD Biosciences) was used to perform dead cell exclusion. SYTO 16 Green Fluorescent Nucleic Acid Stain (ThermoFisher) was used to identify nucleated cells. Large and small macrophages were FACS sorted on a FACSAria III (BD Biosciences) following the gating strategy shown in Fig. S2 .
5
Characterization of Cell Populations in Transplanted Scaffolds
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Transplanted scaffolds were harvested from defect area after EMC and IMC post-implantation for 1 week. The scaffolds were minced and cell were flushed with Ca2+ and Mg2+ free PBS with 2% fetal bovine serum for at least 3 times and then incubated with Anti-CD90 antibody, a surface marker of MSCs, at 1:100 dilution for 30 mins (BD Biosciences). For cell culture, the cells were trypsinized after cultured for 3 days with DMSO or GW4869, and incubated with M2 macrophages antibodies Anti-CD68 (BD Biosciences) and Anti-CD163 (BD Biosciences) at 1:100 dilution for 30 mins. DAPI was used to exclude dead cells. The flow cytometry analysis of positive cells was performed on the Accuri-C6 (BD Bioscience).
6
Phenotypic Profiling of THP-1 Macrophages
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The following fluorescence-conjugated monoclonal mouse anti-human antibodies were used for cell surface staining: anti-CD14, anti-CD163, anti-CD206, anti-CD11b, anti-B7H1, anti-CD40, and anti-HLA-DR (all from BD Biosciences). Isotype-matched negative controls were used throughout the investigations. The THP-1 cells (1 3 10 5 cells/well in a 24-well plate) treated with 50% of the control or tumor CM for 72 h were incubated with monoclonal antibodies for 45 min in the dark at 4°C. For intracellular staining (CD68; BD Biosciences), the THP-1 cells were fixed with Cytofix and permeabilized with Perm/Wash buffer (both from BD Biosciences) according to the manufacturer's instructions. The stained cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences) and at least 10,000 events were measured per sample. FlowJo software (FlowJo LLC) was used to analyze the expression of surface markers. All experiments were performed in at least triplicate.
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