Antibodies for flow-cytometric measurements of murine cells were CD90, CD45, CD11b, CD206, CD14, CD36, CD38, CD319, F4/80, CD3, CD4, and CD8 (BD Biosciences), ABCA1 (Novus Biologicals), and CD24 (Biolegend). Antibodies for flow-cytometric measurements of human cells were CD45, CD11b, CD206, CD14, CD36, CD38, CD319 (BD Biosciences), and ABCA1 (Novus Biologicals).
Cd206
Manufactured by BD
Sourced in United States
CD206 is a lab equipment product. It is a receptor that is expressed on the surface of certain types of cells, such as macrophages and dendritic cells. CD206 plays a role in the recognition and uptake of glycoproteins.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b, by BD (12 mentions)
F4 80, by BD (7 mentions)
Facscalibur, by BD (5 mentions)
Cd11b, by BioLegend (4 mentions)
Cd163, by BD (3 mentions)
Hla dr, by BD (3 mentions)
Ifn γ, by BD (3 mentions)
Cd16 32, by BD (3 mentions)
Cd163, by Thermo Fisher Scientific (3 mentions)
Cytoflex flow cytometer, by Beckman Coulter (2 mentions)
Cd206, by Abcam (2 mentions)
Gm csf, by Thermo Fisher Scientific (2 mentions)
M csf, by Thermo Fisher Scientific (2 mentions)
Cd204, by BD (2 mentions)
Cd206, by BioLegend (2 mentions)
Fixable viability dye, by Thermo Fisher Scientific (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Fc block, by BD (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Hla dr, by Thermo Fisher Scientific (2 mentions)
50 protocols using cd206
1
Murine and Human Cell Immunophenotyping
2
Identifying Lung Macrophage Subsets
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The MACS CD206+ enriched population of lung resident macrophages were incubated with FcR Block (422302; BioLegend) for 5 min and stained at a dilution of 1:50 with one of two panels of directly conjugated antibodies for 30 min at 4°C: anti-human CD45 (563792; BD), CD204 (371906; BioLegend), CD206 (321132; BioLegend), CD14 (562698; BD Biosciences), CD16 (302028; BioLegend), ACE2 (FAB933P; R&D), HLA-DR (307618; BioLegend), CD11b (393114; BioLegend), CD11c (301644; BioLegend); anti-human CD45 (324016; BioLegend), CD204 (371904; BioLegend), and CD206 (321103; BioLegend). Stained cells were then washed with FACS buffer (2% FBS in PBS) three times, and then incubated with cell viability marker propidium iodide (PI, 1 μg/ml, 421301; BioLegend). Flow cytometry was performed on a FACS Aria II (BD Biosciences).
Living (PI−) single, immune (CD45+), and lung resident macrophages (CD206+) were stained for the above panel of cell surface antigens that have previously been suggested to segregate them into AMs and IMs, and that were differentially expressed according to the scRNA-seq transcriptomic profiles obtained from lung slice culture and sorted into CD206+CD204hi and CD206+CD204lo populations. The sorted populations were directly subjected to 10x single-cell mRNA sequencing at BSL2 as described above, which confirmed their molecular identities as AMs and IMs, respectively.
Living (PI−) single, immune (CD45+), and lung resident macrophages (CD206+) were stained for the above panel of cell surface antigens that have previously been suggested to segregate them into AMs and IMs, and that were differentially expressed according to the scRNA-seq transcriptomic profiles obtained from lung slice culture and sorted into CD206+CD204hi and CD206+CD204lo populations. The sorted populations were directly subjected to 10x single-cell mRNA sequencing at BSL2 as described above, which confirmed their molecular identities as AMs and IMs, respectively.
3
Determining Immune Cell Phenotypes
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HB8902® cells incubated with PPIs (3 h) were collected with a cell scraper in EMEM. According to the manufacturer’s instructions, 1 µL of anti-human TLR4 (CD274, Cat. Nº 145404, BD Biosciences, San José, CA, USA), CD68 (Cat. Nº. 562117, BD Biosciences) and CD206 (Cat. Nº 555954, BD Pharmingen, San José, CA, USA) antibodies were added to cell suspensions. After gentle vortexing, samples were incubated for 15 min in the dark. Cells were then analyzed using the FACS Diva software (BD Biosciences). At least 10,000 events were analyzed for each independent sample.
4
Cell Surface Marker Expression Analysis
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To measure the cell surface expression of CD47, HER2, EGFR, and CD20, cells were stained with conjugated primary antibodies (BD Biosciences) for 30 minutes at 4°C, washed, and resuspended in staining buffer. Macrophages were incubated with anti-CD16/32 (BD Biosciences) for 15 minutes at room temperature to block nonspecific binding and stained with CD14, CD68, CD80, CD163, CD206, PD-L1, and HLA-DR (BD Biosciences). Cells were acquired using a BD LSR X-20, and the data were analyzed using FlowJo software.
5
Endocytic receptor and immune cell analysis
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Endocytic receptors were stained using the following antibodies: CD206/MR (eBioscience), DC-SIGN, CD36 (both from BD Biosciences), SR-A1 (R&D Systems), LOX-1 (BioLegend), and SR-B1 (Novus Biologicals). DC and T cell phenotypes were examined using antibodies against the following markers: HLA-ABC (BioLegend), CD206, CD40, CD80, CD83, IL-2 (BD Biosciences), CD4, CD8, TNF-α, IL-2, and HLA-DR (Beckman Coulter). Data were acquired with an Accuri C6 cytometer (BD Biosciences) and analyzed using CFlow Plus software.
6
Macrophage Polarization Assay
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After 1.5 × 105 RAW264.7 seeded in a 6-well plate for 12 h, 10 ng/mL lipopolysaccharide (LPS; Beyotime, China) was used to activate M1 phenotypes from M0 macrophages for 8 h. Then the culture medium was refreshed with DMEM extracts of SG, 15SrP/SG, Rg1/SG, and Rg1/15SrP/SG scaffolds. The LPS-treated cells were fixed in 4% PFA solution and stained with FITC-phalloidin (ATT Bioquest, USA) for 1 h and DAPI (thermo fisher, USA) for 5 min. The stained cells were captured by confocal laser scanning microscope (Zeiss, German). After cultured with the scaffold extracts for 3 days, the cells were digested with trypsin solution, washed, and resuspended in 1% BSA/PBS with CD86 (Thermo Fisher) and CD206 (Thermo Fisher) antibodies for 30 min on ice. After washing again, the cell suspensions were performed on a flow cytometry (BD, USA), and the types of macrophages (M1: CD86; M2: CD206) were analyzed.
7
Intestinal Macrophage Phenotyping and Activation
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The presence of MΦ in the intestinal lamina propria of CDA or HC subjects and the phenotype of monocytes derived MΦ was evaluated by flow cytometry as follow: MΦ were stained with antibodies anti-CD80 (BD bioscience) and CD206 (BD bioscience) to evaluate the percentages of classically activated pro-inflammatory (M1) and alternatively activated anti-inflammatory (M2) MΦ, respectively; CD 68 antibody was used as a pan marker for MΦ. Activation of IFNγR and pSTAT1 on monocytes derived MΦ was evaluated using IFNγR (Thermofisher) and intracellular pSTAT1 (Ser767) (Abcam) antibodies after methanol permeabilization. Data were acquired with BD FACS Calibur flow cytometer instrument and analyzed with BD CellQuestPro and FlowJo softwares.
8
Quantifying Macrophage Phenotypes by Flow Cytometry
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The percentages of macrophages with the M1 or M2 phenotype were analysed by flow cytometry. Briefly, cultured peritoneal macrophages with Gm26917 knockdown or LPS treatment were harvested, blocked using Fc receptor blocker, and then incubated with specific antibodies for 30 min on ice. Macrophages were identified as F4/80-positive cells. The percentages of inflammatory M1 macrophages and regulatory M2 macrophages were then evaluated using antibodies against the M1 macrophage marker Ly6C (BD Pharmingen) and M2 macrophage marker CD206 (BD Pharmingen), respectively. All samples were acquired and analyzed using a CytoFLEX flow cytometer (Beckman Coulter) with CytExpert software (version 2.4).
9
Characterization of Tumor-associated Macrophages
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The induced M2 macrophages were probed with F4/80 (1:500), CD86 (1:500), CD206 (1:200), or isotype immunoglobulin G 2b (1:300, all from Abcam). Then, the M2 macrophages (1 × 106 cells/mL) were detected on a flow cytometer (FACSCalibur, BD Biosciences) for measuring the proportion of CD86+ and CD206+ cells. M2 macrophages were F4/80+ and CD206+. Cell apoptosis was tested by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Invitrogen). PANC-1 and AsPC-1 cells, and P-CSCs and A-CSCs were added with dimethyl sulfoxide (10 μmol/mL) or anti-tumor drug perifosine (10 μmol/mL) (Zhou et al. 2020 (link)). Treated for 48 h, cells were fixed in 70% ethanol, adjusted to 1 × 106 cells/mL with 1× Binding Buffer, and stained by 5 μL Annexin V-FITC and 1 μL PI (Invitrogen). The flow cytometer (FACSCalibur) was adopted to apoptosis detection.
10
Cellular Responses to Immunomodulatory Stimuli
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Lipopolysaccharide (LPS) (Sigma, St. Louise, MO, USA), M-CSF and GM-CSF (PeproTech, Rocky Hill, NJ, USA) were applied at final concentrations of 50 ng/ml. Recombinant human cytokines IL-4, IL-10, IL-13, IFNγ and TGF-β (R&D Systems, Minneapolis, MN, USA) were applied at final concentrations of 20 ng/ml. Antibodies against CD86, CD273, CD274, CD14, CD206 (all BD Bioscience-Pharmingen, San Diego, CA, USA), HLA-DR (eBioscience, San Diego, CA, USA) and isotype control mouse IgG2a (BD Bioscience-Pharmingen and eBioscience), mouse IgG2b (BD Bioscience-Pharmingen), were used for flow cytometric analyses. Dextran Alexa Fluor 647, 10,000 MW, anionic (Life Technologies, Stockholm, Sweden) was used for endocytosis experiments at 10 μg/ml.
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