Cd206 pecy7

Manufactured by Thermo Fisher Scientific

CD206-PeCy7 is a flow cytometry reagent that detects the expression of the CD206 receptor, also known as the macrophage mannose receptor, on the surface of cells. CD206 is a type I transmembrane protein that is involved in the endocytosis of glycoproteins. The PeCy7 fluorochrome is conjugated to the CD206 antibody, enabling the visualization and quantification of CD206-expressing cells using flow cytometry.

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CD206 (71 citations)

5 protocols using cd206 pecy7

Plasmid-based PTEN Gene Therapy

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The three plasmid systems, including target gene plasmid (pAAV-CAG-mCherry), capsid plasmid (pAAV-6, pAAV-1, pAAV-7m8, pAAV-7, pAAV-8, pAAV-10) and helper plasmid (pHelper), were all purchased from Fenghui Biotechnology (Hunan, China). The complementary DNA (cDNA) encoding the mouse PTEN gene was synthesized from GENEWIZ (Suzhou, China). pAAV-CAG-PTEN was obtained by replacing mCherry gene in pAAV-CAG-mCherry with PTEN gene. Reverse the PTEN gene sequence in pAAV-CAG-PTEN to get pAAV-CAG-PTEN^R.
The anti-PD-1 used in this study is AUNP-12, a polypeptide purchased from Selleck (USA) with a purity of 99.20%. Cy5-AUNP-12 was purchased from Guoping pharmaceutical co (Anhui, China) with a purity of 90%. CpG was synthesized in GENEWIZ (Suzhou, China) with 90% purity.
Antibodies for Western blot (WB) and Immunofluorescence (IF): anti-mouse PTEN, CRT, β-actin and anti-rabbit FITC, CY3 fluorescent secondary antibodies were purchased from ABclonal Tech (Wuhan, China). Antibodies for flow cytometry test: CD11c-PE, CD80-FITC, CD86-APC, CD3-APC, CD8-FITC, CD4-APC-eflour780, CD69-PE, CD274-PE, CD11b-FITC, CD206-PE-Cy7, CD86-APC, IL-4-PE, IFN-γ-APC, TNF-α-eflour450, IL-2-PE5.5, CD4-APC-eflour780, CD44-APC, CD62L-PE were all purchased from Invitrogen (USA).

Zhang Y., Yang L., Ou Y., Hu R., Du G., Luo S., Wu F., Wang H., Xie Z., Zhang Y., He C., Ma C., Gong T., Zhang L., Zhang Z, & Sun X. (2023). Combination of AAV-delivered tumor suppressor PTEN with anti-PD-1 loaded depot gel for enhanced antitumor immunity. Acta Pharmaceutica Sinica. B, 14(1), 350-364.

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Isolation and Analysis of Mouse Skin Immune Cells

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Mouse skin samples were mechanically disrupted followed by digestion using Liberase TL (Sigma, 1 h incubation at 37 °C) at concentration 0.25 mg/mL dissolved in RPMI‐1640 with l‐glutamine (Corning), supplemented with 1 mM sodium pyruvate, MEM nonessential amino acids, and 20 mM HEPES (all from Gibco). Following digestion, the cell suspension was filtered twice consequently using 70‐ and 40‐μm strainers (Greiner), and washed and stained for analysis in flow buffer containing 2% bovine serum albumin (Sigma). The following anti‐mouse antibodies were used: F4/80 APC, CD45 eFluor^® 450, Ly6c Alexa Fluor^® 488, CD206 PE‐Cy7, and Fixable Viability Dye eFluor 506 (all from Invitrogen and Thermo Fisher Scientific). Appropriate isotype controls were also purchased from Invitrogen, Thermo Fisher Scientific, and TruStain FcX, and Fc blocking antibodies were from Biolegend. Data were collected using BD FACSCanto^® II cytometer and analyzed with FlowJo v10.5.3 software (BD).

Biyashev D., Onay U.V., Dalal P., Demczuk M., Evans S., Techner J, & Lu K.Q. (2020). A novel treatment for skin repair using a combination of spironolactone and vitamin D3. Annals of the New York Academy of Sciences, 1480(1), 170-182.

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In Vivo Uptake of Tumor Cells

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C57BL/6 female mice (6–9 weeks old) were injected intra-peritoneum with PBS containing 3 × 10⁶ B16-F10 cells (BNIP3^WT or BNIP3^KD) pHrodo-labelled (Life Technologies, P36600). The peritoneal cells were collected 24 h post-injection via peritoneal lavage with Ca²⁺- and Mg²⁺-free PBS. The cells were transferred to an ultra-low attachment V-bottom plate and stained with the fixable Live/Dead Yellow stain (Invitrogen). After blocking the F_c receptor (CD16/32, eBioscience, 16-0161-82), cells were stained for F4/80-eFluor780 (eBioscience, 47-4801-80), CD11b-eFluor450 (eBioscience, 48-0112-82), MHCII-FITC (eBioscience, 11-5321-81), CD86-APC (eBioscience, 17-0862-81), CD206-PeCy7 (eBioscience, 25-2061-80) in FC buffer. Samples were acquired on a Gallios^™ flow cytometer and the data analysis was performed via FlowJo_V10^™ software.

Romano E., Rufo N., Korf H., Mathieu C., Garg A.D, & Agostinis P. (2018). BNIP3 modulates the interface between B16-F10 melanoma cells and immune cells. Oncotarget, 9(25), 17631-17644.

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Phenotyping Macrophages and Murine Immune Infiltrates

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10,000 cells per sample were collected on a FACSCalibur instrument (BD Biosciences, San Jose, CA) using Cell Quest Software (BD Bioscience) or a BD Canto II Instrument (BD Biosciences) using FACSDIVA software (BD Biosciences) and analyzed using FlowJo v10 (FlowJo LLC, Ashland, OR). For phenotyping macrophages, the following monoclonal antibodies (MAbs) were used: CD14-PerCP (BD), CD33-PE (BD), CD80-FITC (BD Biosciences), and CD163-AlexaFluor647 (Biolegend, San Diego, CA). For detection of VV transgenes, the following MAbs were used: AffiniPure F(ab’)2 Fragment Goat Anti-Human IgG: AlexaFluor 647 (Jackson ImmunoResearch Laboratories, West Grove, PA), human CD47-APC (eBioscience), murine CD47-APC (eBioscience). For murine immune infiltrate studies, the following MAbs were used: CD3-APC (BD Biosciences), CD11b-AlexaFluor 488 (BD Biosciences), CD11c-PerCP-Cy5 (BD Biosciences), CD206-PECy7 (eBioscience), F4/80-PE (Biolegend), Ly6G-AlexaFluor 700 (BD). Isotype-matched controls (BD) were used for each fluorophore.

Cao F., Nguyen P., Hong B., DeRenzo C., Rainusso N.C., Rodriguez Cruz T., Wu M.F., Liu H., Song X.T., Suzuki M., Wang L.L., Yustein J.T, & Gottschalk S. (2020). Engineering Oncolytic Vaccinia Virus to redirect Macrophages to Tumor Cells. Advances in cell and gene therapy, 4(2), e99.

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Multiparametric Immune Profiling of Tumor

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Single-cell suspensions derived from the tumor, LN, spleen, blood or abdominal cavity were harvested and stained with fluorochrome-conjugated antibodies. However, the tumor single-cell suspensions were stained with Fixable Viability Stain 510 (Thermo Fisher Scientific) prior to antibody staining to distinguish between living and dead cells. To analyze M2 macrophages, cells were stained with anti-CD45 PerCP/Cy5.5 (BioLegend), F4/80 APC (BioLegend), CD11b PE (BioLegend) and CD206 PE/CY7 (eBioscience) antibodies. To detect CD8⁺CD122⁺ Tregs, cells were stained with anti-CD45 PerCP/Cy5.5, CD8 APC/eFluor780 (eBioscience) and CD122 PE or isotypes (BioLegend) antibodies. To measure IFN-γ⁺ T cells, 1 × 10⁶ cells were incubated in 96-well plates in the presence of PMA (50 ng/ml, MultiSciences) and Ionomycin (1 μg/ml, MultiSciences) for 6 h at 37°C. Monensin (5 μg/ml, MultiSciences) was added 2 h after the addition of PMA/Ionomycin. Cells were first stained with anti-CD45 PerCP/Cy5.5 and CD8 APC/eFluor780, fixed in 1% paraformaldehyde, permeabilized and then stained with anti-IFN-γ APC (eBioscience). Cells finally were analyzed through FACSCalibur (BD Biosciences).

Zhang Q., Huang H., Zheng F., Liu H., Qiu F., Chen Y., Liang C.L, & Dai Z. (2020). Resveratrol exerts antitumor effects by downregulating CD8+CD122+ Tregs in murine hepatocellular carcinoma. Oncoimmunology, 9(1), 1829346.

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