Anti ccr7

Manufactured by Thermo Fisher Scientific

Sourced in United States

Anti-CCR7 is a reagent used in flow cytometry applications. It is a monoclonal antibody that binds to the CCR7 receptor, which is expressed on the surface of certain immune cells. The primary function of Anti-CCR7 is to facilitate the identification and analysis of CCR7-positive cells in a sample.

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Anti-CCR7 PE (5 citations) Anti-CCR7-APC (4 citations) PE-conjugated anti-mouse CD40/MHC-II/CCR7/Foxp3/PD-L1 (2 citations) Anti-CCR7 (4B12, (2 citations) PE anti-mouse CCR7 (2 citations) Anti-mouse CD197 (CCR7)-PE-Cy7 (2 citations)

6 protocols using anti ccr7

Comprehensive Immune Cell Profiling by Flow Cytometry

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The following antibodies were used for flow cytometry analysis: anti-IFNγ (XMG1.2, 1:50), anti-CD45.1 (A20, 1:100), anti-CD45.2 (104, 1:100), anti-IL1β-APC (NJTEN3, 1:50), anti-CD44 (IM7, 1:100), anti-CD62l (MEL-14, 1:100), anti-F4/80 (BM8, 1:100), anti-CCR7 (4B12, 1:100), anti-CD4 (RM4-5, 1:200), anti-CD8α (53-6.7, 1:200), anti-H-2K^b (AF6-88.5.5.3, 1:200), anti-Ly6G (RB6-8C5, 1:100), anti-MHC class II (I-A/I-E) (M5/114.15.2, 1:100) and anti-CD11c (N418, 1:100) were from eBioscience. Anti-CD103 (2E7, 1:100) was from Biolegend. Annexin V apoptosis Detection kit (556547) was from BD Pharmingen. For intracellular cytokine staining, Cytofix/Cytoperm plus kit (BD, cat. 55508) was used. For Foxp3 staining, Foxp3 staining buffer set (eBioscience, cat. 00-5523-00) was used. Multiple-colour flow cytometric analysis was performed using FACSAria (BD Biosciences; Franklin Lakes, NJ, USA). For FACS sorting, cells stained with FACS
antibodies were sorted on BD FACSAria device. FlowJo software was used for data acquiring and analysis.

Yao Y., Chen S., Cao M., Fan X., Yang T., Huang Y., Song X., Li Y., Ye L., Shen N., Shi Y., Li X., Wang F, & Qian Y. (2017). Antigen-specific CD8+ T cell feedback activates NLRP3 inflammasome in antigen-presenting cells through perforin. Nature Communications, 8, 15402.

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Isolation and Proliferation of Human CD4+ T Cells

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Buffy coats were obtained from healthy donors who gave informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated using Lympholyte (Cederlane, CL5020) density gradient separation. CD4⁺ T cells were MACsorted from PBMCs using human anti‐CD4 magnetic beads (Miltenyi Biotech, 130–045–101), stained with carboxyfluorescein succinimidyl ester (CFSE; Life Technologies, C34554). and plated in RPMI Medium 1640 (Gibco, A10491‐01) supplemented with 10% FBS, 1% penicillin/streptomycin, 1% Minimum Essential Medium Nonessential Amino Acid (MEM‐NAA; Gibco, 11140–035) and 0.1% 2‐mercaptoethanol. For stimulation, T cells were incubated with anti‐CD3/anti‐CD28 beads (Dynabeads Human T‐Activator, Gibco, 11131D) for 5 days. T cell proliferation was measured by flow cytometric CFSE dilution assay using LSRII Fortessa analyzer. T cell phenotype was assessed using anti‐CD45RA, anti‐CD45RO, anti‐CCR7, and anti‐CD27 antibodies (all from eBioscience).

Vdovenko D., Balbi C., Di Silvestre D., Passignani G., Puspitasari Y.M., Zarak‐Crnkovic M., Mauri P., Camici G.G., Lüscher T.F., Eriksson U, & Vassalli G. (2022). Microvesicles released from activated CD4+ T cells alter microvascular endothelial cell function. European Journal of Clinical Investigation, 52(6), e13769.

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Multicolor Flow Cytometry Panel for Immune Profiling

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Cells were stained with the following antibodies obtained from BD Biosciences (Franklin Lakes, NJ, USA), eBioscience, or BioLegend (San Diego, CA, USA): anti-CD4 (BioLegend, 100451), anti-CXCR3 (eBioscience, 12-1831-82), anti-CCR7 (eBioscience, 12-1971-82), anti-CD44 (eBioscience, 12-0441-83), anti-CD62L (BioLegend, 104406), anti-CD11c (BD Bioscience, 553801), anti-MHCII (BioLegend, 107631), anti-CD80 (BD Biosciences, 553769), anti-CD86 (BD Biosciences, 553692), anti-B220 (eBioscience, 12-0452-83), anti-CD8 (BD Bioscience, 553032), anti-CD103 (BD Bioscience, 557495), anti-CD45 (BioLegend, 103132), and anti-CD11b (BioLegend, 101263). For Th1 and Treg analyses, cells were stained for surface markers, permeabilized with the Intracellular Fixation and Permeabilization Buffer Set (eBioscience, 88-8824-00), and then stained with anti-IFN-γ (BioLegend, 505825) and anti-FOXP3 (eBioscience, 17-5773-82) antibodies. The following antibodies were obtained from Cell Signaling Technology (Danvers, Massachusetts, USA): anti-Ezh2 (#5246), anti-Runx1 (#4336), and anti-H3K27me3 (#9733). The Alexa Fluor™ 488 Goat Anti-Rabbit SFX Kit from Invitrogen (Carlsbad, CA, USA) was used as a secondary antibody. Multicolor flow cytometric analysis was performed using a CytoFLEX LX (Beckman Coulter, Indianapolis, IN, USA).

Wang Y., Wang Q., Wang B., Gu Y., Yu H., Yang W., Ren X., Qian F., Zhao X., Xiao Y., Zhang Y., Jin M, & Zhu M. (2020). Inhibition of EZH2 ameliorates bacteria-induced liver injury by repressing RUNX1 in dendritic cells. Cell Death & Disease, 11(11), 1024.

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Characterization of Senescent and Exhausted T Cells

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PBMCs were isolated using Ficoll-Paque (GE Healthcare, USA), and the cellular phenotypic of senescent and exhausted T cells was analyzed by flow cytometry. For surface markers analysis, cells were stained in PBS containing 2% fetal bovine serum (FBS, Thermo fisher, USA) with antibodies as indicated. Then, flow cytometric analysis was carried out in BD LSRFortessa X20. The gating strategy to identify T cell subsets was applied as described previously (27 (link)). Antibodies used in this study were purchased from BD Biosciences and eBioscience, including anti-CD4 (GK1.5, 1:100), anti-CD8 (53-6.7, 1:100), anti-CD25 (PC61, 1:100), anti-CD45RA (HI100, 1:100), anti-CXCR3(G025H7, 1:100), anti-CCR4 (L291H4, 1:100), anti-CCR6 (G034E3, 1:100), anti-CCR7 (G043H7, 1:100), anti-CD127 (A019D5, 1:100), anti-CXCR5 (RF8B2, 1:100), anti-CD28 (CD28.2, 1:100), anti-CD57 (NK-1, 1:100), anti-KLRG1 (2F1, 1:100), anti-PD-1 (EH12.2H7, 1:100), anti-TIM3 (F38-2E2, 1:100), and fixable viability dye eFluor 780 (eBioscience, Cat#65-0865-14, 1:1,000).

Tang X., Deng B., Zang A., He X., Zhou Y., Wang D., Li D., Dai X., Chen J., Zhang X., Liu Y., Xu Y., Chen J., Zheng W., Zhang L., Gao C., Yang H., Li B, & Wang X. (2022). Characterization of age-related immune features after autologous NK cell infusion: Protocol for an open-label and randomized controlled trial. Frontiers in Immunology, 13, 940577.

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Isolation and Sorting of CD8+ T Cell Subsets

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Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors by density gradient centrifugation with Lymphoprep (AXIS-SHIED Rodelokka, Oslo, Norway). CD3⁺ and CD8⁺ lymphocytes were isolated by a magnetic-activated cell sorter (MACS) using CD3 Microbeads and a CD8 T cell isolation kit (MiltenyiBiotec), respectively. The cells were then labeled with anti-CD8, anti-CCR7, and anti-CD45RA (Ebiosciences) and sorted into naïve and memory CD8⁺ T cell subsets on a BD fluorescence activated cell sorting (FACS) Aria sorter (BD Biosciences). For extraction of TILs, tissue from lung tumor samples were minced and digested with collagenase (2.5 mg/ml collagenase I) at 37 °C for one hour. Cell suspension was then twice filtered through 100-μm and 40-μm cell strainers (BD Biosciences) to obtain single cells. TILs were isolated from single cells utilizing density gradient centrifugation with Lymphoprep and further purified using CD8 MicroBeads (MiltenyiBiotec).

Nguyen H.H., Kim T., Song S.Y., Park S., Cho H.H., Jung S.H., Ahn J.S., Kim H.J., Lee J.J., Kim H.O., Cho J.H, & Yang D.H. (2016). Naïve CD8+ T cell derived tumor-specific cytotoxic effectors as a potential remedy for overcoming TGF-β immunosuppression in the tumor microenvironment. Scientific Reports, 6, 28208.

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Phenotypic Analysis of Senescent and Exhausted T Cells

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Peripheral blood mononuclear cells (PBMCs) were isolated using coll-paque (GE Healthcare, USA) for cellular phenotypic analysis of senescent and exhausted T cells by ow cytometry. For analysis of surface markers, cells were stained in PBS containing 2% fetal bovine serum (FBS, Thermo sher, USA)
with antibodies as indicated. Then ow cytometric analysis was carried out in BD LSRFortessa X20. The gating strategy to identify T cell subsets was applied as described previously 28 . Brie y, markers related to T cell senescence and exhaustion were also monitored using PD-1 TIM3 CD28 and CD57 antibodies.
Antibodies used in this study were from BD Biosciences and eBioscience, including anti-CD4 (GK1.5, 1:100), anti-CD8 (53-6.7, 1:100), anti-CD25 (PC61, 1:100), anti-CD45RA (HI100, 1:100), anti-CXCR3 (G025H7, 1:100), anti-CCR4 (L291H4, 1:100), anti-CCR6 (G034E3, 1:100), anti-CCR7 (G043H7, 1:100), anti-CD127 (A019D5, 1:100), anti-CXCR5 (RF8B2, 1:100), anti-CD28 (CD28.2, 1:100), anti-CD57 (NK-1, 1:100), anti-KLRG1 (2F1, 1:100), anti-PD-1 (EH12.2H7, 1:100), anti-TIM3 (F38-2E2, 1:100).

Tang X., Deng B., Zang A., He X., Zhou Y., Wang D., Li D., Zhang X., Liu Y., Yong-hua X., Chen J., Zheng W., Zhang L., Gao C., Yang H., Li B., & Wang L. (2021). Characterization Of Age-Related Immune Features After Autologous NK Cell Infusion.

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