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 °C. 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 flow cytometer (BD Biosciences). Flow cytometric analysis was performed on FlowJo software (FlowJo, Ashland, OR, USA).
Cd206 apc
Manufactured by Thermo Fisher Scientific
Sourced in United States
CD206-APC is a fluorescent-labeled antibody targeting the CD206 antigen. CD206, also known as the macrophage mannose receptor, is a cell surface receptor expressed on various immune cells, including macrophages and dendritic cells. The APC (Allophycocyanin) fluorescent label allows for the detection and analysis of CD206-expressing cells using flow cytometry or other fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
F4 80 fitc, by Thermo Fisher Scientific (6 mentions)
F4 80 pe, by Thermo Fisher Scientific (6 mentions)
Inos pe, by Thermo Fisher Scientific (4 mentions)
Cd86 pe, by Thermo Fisher Scientific (4 mentions)
Facsaria flow cytometer, by BD (3 mentions)
Cd206 pe, by Thermo Fisher Scientific (3 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (3 mentions)
Cd11b fitc, by Thermo Fisher Scientific (3 mentions)
Facscalibur, by BD (3 mentions)
Ly6c apc, by Thermo Fisher Scientific (2 mentions)
Cd11b pe, by Thermo Fisher Scientific (2 mentions)
Cd11b apc, by Thermo Fisher Scientific (2 mentions)
Cd45 fitc, by Thermo Fisher Scientific (2 mentions)
Cd8 apc, by Thermo Fisher Scientific (2 mentions)
Cd45 efluor450, by Thermo Fisher Scientific (2 mentions)
Cd11b apc cy7, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Cd11b fitc, by BD (2 mentions)
Cd4 apc, by Thermo Fisher Scientific (2 mentions)
35 protocols using cd206 apc
1
Macrophage Phenotypes in Colorectal Cancer
2
Multicolor Flow Cytometry of Blood and Tissue
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Peripheral blood samples were collected into heparinized tubes. The left ventricle tissues of peri-infarct area were collected in cold staining buffer (2% FBS, 0.05% NaN3 in PBS). For tissue cytometry analysis, single-cell suspensions of tissues were obtained using gentleMACS™ Dissociator (Miltenyi Biotec). Samples were stained with fluorochrome-conjugated antibodies against extracellular or intracellular markers according to the manufacturer’s protocols. The following antibodies were used for extracellular staining: CD11b-FITC (BD Bioscience), F4/80-PerCP/Cy5.5 (eBioscience) and Ly6C-APC (eBioscience). The intracellular fixation and permeabilization kit (eBioscience) was used according to the manufacturer’s protocol for intracellular staining. Cells were then washed with staining buffer and stained with CD206-APC (eBioscience) and iNOS-PE (eBioscience). After incubation with antibodies, blood samples were treated with red blood cell lysis buffer to remove red blood cells and heart tissue samples were filtered through a 70-μM filter. Flow cytometry was performed using FACS Aria flow cytometer (BD Bioscience), and flow cytometry data were analyzed using FlowJo software (TreeStar, Ashland, OR).
3
Flow Cytometry Analysis of Tumor Immune Cells
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Tumor tissue was digested in a 0.3% collagenase/0.1% hyaluronidase solution, pressed through a nylon mesh filter to obtain a single cell suspension and incubated in red cell lysis buffer (0.17 M Tris-HCl, 0.16 M NH4Cl) for 3 min, spun down, and resuspended in FACS buffer (PBS + 1.5% FBS). Equal numbers of cells were stained with a viability dye and combinations of the following antibodies: CD8a-PECy7 (eBioscience, San Diego, CA, USA, 25-0081-82), F4/80-PECy7(eBioscience, 25-4801-82), CD206-FITC (Biolegend, 141704), Ly6G-APC (eBioscience, 17-5931-81), CD11b-PE (eBioscience, 12-0112-82), CD45-PE-Cy5 (eBioscience, 15-0451-83), IFN-γ-APC (eBioscience, 17-7311-82), and SIINFEKL pentamer-PE (ProImmune, Oxford, UK). For autophagy analysis using Cyto-ID staining of bone marrow derived macrophages, cells were first stained for surface markers (CD80-PE-Cy5 (eBioscience, 15-0801-81) and CD206-APC (eBioscience, 17-2061-82)) followed by staining with Cyto-ID for 30 min at 37 °C, per the manufacturer’s protocol (Enzo Life Sciences, Farmingdale, NY, USA, ENZ-51031). Flow cytometric data were acquired on a BD FACSCanto II cytometer and analyzed using FACSDiva software version 9.2 (BD Biosciences, San Jose, CA, USA).
4
Flow Cytometry Analysis of Macrophage Phenotype
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To evaluate cell viability, cells were detached from plates using fresh EDTA (5 mM) in PBS, pooled with cell supernatants containing propidium iodide (PI, 10 μg/mL) and recorded using an LSR II flow cytometer (Becton Dickinson, NJ). PI exclusion analysis for each sample was performed with 10,000 single cell, non-debris events using a FSC-A versus PI FACS plot. For cell surface marker expression analysis, stimulated BMDMs were harvested as described above and incubated with the following antibodies: F4/80-FITC (1:200, eBioscience; clone BM8), MHCII-AF700 (1:200, eBiocience; M5/114.15.2), CD206-APC (1:200, eBioscience; MR6F3) and CD11b-PE-Cy7 (1:400, eBioscience; M1/70) for 30 min on ice. Cells were washed once in PBS prior to analysis by flow cytometry. Analysis of cell surface marker expression was performed on 10,000 single cell, PI negative, gated events. Flow cytometry data were analyzed using WEASEL version 2.7 software (Frank Battye).
5
Macrophage Polarization and Cell Death
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RAW264.7 cells were left alone or cultured with IL-4 (5 ng/ml) for 48 h. Some of the cells were also treated with 0.1 μM microcystin-LR. Cells were harvested and washed twice with 3 ml 0.5% BSA-PBS by centrifuging at 500 g for 5 min at 4 °C. The cell pellet was suspended in 1 ml 0.5% BSA-PBS and the total number was calculated by a cell counter. Following preincubation with mouse Fc block (eBioscience, San Diego, California, USA) for 15 min, the cell suspension was stained with anti-mouse CD11b-FITC (eBioscience) and CD206-APC (eBioscience) at 4 °C for 30 min in the dark. After washing and resuspending in PBS, the cells were analyzed by a flow cytometer (FACSCalibur, BD Biosciences, San Jose, California, USA). For PI and Annexin-V double staining, cells were suspended in 100 μl of a buffer solution (10 mM HEPES/NaOH, 140 mM NaCl, 2.5 mM CaCl2, 5 mM KCl, pH 7.4) and stained with FITC-conjugated AnnexinV and PI (50 μg/ml) for 30 min at room temperature in the dark.
6
Multiparametric Flow Cytometry Analysis
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Cells were collected after different treatments and then made into single-cell suspensions. Next, cell suspension was incubated with F4/80-FITC (11-4801-82, eBioscience, San Diego, CA, USA), iNOS-PE (61-5920-82, eBioscience), and CD206-APC (17-2061-82, eBioscience). After incubation, cells were collected and analyzed using a CytoFlex flow cytometer (Beckman Coulter, Brea, CA, USA). Data were analyzed using the FlowJo software (version 7.0; FlowJo LLC, Ashland, OR, USA).
7
Macrophage Polarization and Insulin Signaling
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All reagents were obtained from Sigma–Aldrich (St. Louis, MO) unless otherwise noted. IFNγ and IL-4 were obtained from R&D Systems (Minneapolis, MN). Lipopolysaccharide (LPS, Sigma E. coli L4391) was diluted in sterile PBS at a final concentration of 1 mg/mL. Novolin® human insulin was purchased from Novo Nordisk (Plainsboro, NJ). Antibodies were purchased from the following sources: F4/80 (AbD Serotec/BioRad, Hercules, CA); CD16/32 (Fc Block, BioLegend, San Diego, CA), CD45-FITC, F4/80-PE, Ly6G/C-PE-Cy7, CD11b-APC, CD11c-APC-eFluor 780, CD11c-eFluor 450, CD206-APC (eBioscience, San Diego, CA), PhosphoAKT-Ser473 and total AKT (Cell Signaling Technology), and insulin (H-86; Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
8
Immune profiling of tumor tissues
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Primary tumor tissues or spleens were dissociated using a gentleMACS Dissociator (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany) after enzyme digestion in serum-free RPMI 1640 medium containing DNase I (1 U/mL; Roche, Basel, Switzerland) and collagenase D (1 mg/mL; Roche) for 20 min at 37 °C with gentle agitation. After single-cell dissociation, the cells were filtered through a 40 μm cell strainer, and the red blood cells were eliminated using BD Pharm Lyse buffer (BD Biosciences, San Jose, CA, USA). The cells were stained by incubating at 4 °C for 1 h using the following antibodies: CD45-FITC, F4/80-PE, CD206-APC, CD86-PE/Cy7, CD8-APC, PD-1-PE, and IFN-γ-PE/Cy7 (e-Bioscience, CA, USA). After staining, the cells were analyzed using a BD FACSLyric flow cytometry system (BD Biosciences) and FlowJo software (Treestar, San Carlos, CA, USA).
9
Tumor Immune Cell Profiling Protocol
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The following antibodies were used for the analysis of tumor infiltration by immune cells: CD45 -eFluor 450 (eBioscience 48-0451-82), CD11b-APC cy7 (eBioscience 47-0112-82), F4/80-PE (eBioscience 12-4801-82), CD8-PE cy7 (eBioscience 25-0081-82), CD4-FITC (eBioscience 11-0042-85), IFNgama-APC (eBioscience 17-7311-82), CD206-APC (eBioscience 17-2061-82). Single-cell suspensions were prepared from the tumor tissues after homogenization or from culture suspension cells. For staining, 100 μl of single-cell suspensions were incubated with antibodies for 30 min. Individual single-color controls were prepared for compensation adjustment. After staining, the samples were washed twice with PBS and resuspended in 600 μl PBS. Flow cytometry data were acquired using a CytoFLEX LX system (Beckman Coulter). FlowJo version 10.0 was used for data analysis. The absolute numbers of CD8+ T cell or macrophage numbers per gram of tumor were the corresponding cell counts from 60 μl of cell suspension (10% of total volume) divided by the tumor weight. The gating strategy is described in each figure’s caption. An example of the gating strategy is given in Supplementary Figure S1 .
10
Flow Cytometric Phenotyping of Liver Cells
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For flow cytometry analysis of tissues, liver cells were obtained based on methods described by Nemeth et al. Briefly, freshly removed liver tissues were minced into small pieces and incubated in RPMI 1640 medium with 300 U/mL collagenase type I (Sigma), 300 U/mL collagenase type IV (Sigma), and 50 U/mL DNase I (Sigma). After incubation, cell suspensions were filtered through a 70-µm cell strainer and then washed with complete RPMI medium. Single-cell suspensions were incubated with an Fc receptor blocker (CD16/32, eBioscience) to reduce nonspecific antibody binding. The panel of antibodies used in these experiments included CD11b-APC, F4/80-FITC, CD206 PE, and iNOS-PE (all from eBioscience). Among these antibodies, CD11b and F4/80 cause cell surface staining, while CD206 and iNOS result in both cell surface and intracellular staining. Cells were then washed and stained with iNOS-PE (eBioscience) and CD206-APC (eBioscience). Flow cytometry was performed using a FACSAria flow cytometer (BD Bioscience), and data were analysed with FlowJo software (TreeStar, Ashland, OR).
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