Anti ccr7 pe

Manufactured by BioLegend

Sourced in United States

Anti-CCR7-PE is a monoclonal antibody that binds to the CCR7 protein. CCR7 is a chemokine receptor that plays a role in the trafficking and homing of T cells and dendritic cells. This antibody is conjugated to the fluorescent dye phycoerythrin (PE) for detection and analysis purposes.

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Close spelling variants of this product

Anti-CCR7-PE-Cy7 PE-conjugated anti-CCR7 PE labeled anti-CCR7 Anti-CD197 (CCR7) PE CCR7-PE Anti-CCR7

4 protocols using anti ccr7 pe

Phenotypic Characterization of Activated T Cells

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PBMC were purified as indicated above and activated with TransAct™ (1:100, Miltenyi-Biotec 130-111-160) and recombinant human IL2 (100 IU/ml; PeproTech). Then at days 0 and days 2-4-6 post-stimulation, the cells were stained with Fixable Viability Stain 450 (250 ng/mL; BD Biosciences), next with anti-CD3-PE-Cy5 (1/30; clone HIT3a; BD Biosciences), anti-CD4-PE-Vio770 (1/100; clone rea623; Miltenyi Biotec), anti-CD8-APC-Cy7 (1/30; clone RPA-T8; Biolegend), anti-CD25-Alexa fluor 488 (1/30; clone M-A251; Biolegend), anti-CD45RA-APC (1/30; clone HI100; Biolegend), anti-CCR7-PE (1/50; clone G043H7; Biolegend), anti-Granzyme-B-PE (1/50; clone GB12; Invitrogen), anti-PD1-Alexa 488 (1/50; clone EH12-2H7; Biolegend) and anti-KLRG1-APC (1/100; clone Rea261; Miltenyi Biotec). FACS analyses were performed on a MACSQuant Analyzer (Miltenyi Biotec) and data analyzed using FlowJo 10 software (FlowJo, LLC). All samples were gated on forward and side scatter, for singlets and for live cells.

Cuche C., Mastrogiovanni M., Juzans M., Laude H., Ungeheuer M.N., Krentzel D., Gariboldi M.I., Scott-Algara D., Madec M., Goyard S., Floch C., Chauveau-Le Friec G., Lafaye P., Renaudat C., Le Bidan M., Micallef C., Schmutz S., Mella S., Novault S., Hasan M., Duffy D., Di Bartolo V, & Alcover A. (2023). T cell migration and effector function differences in familial adenomatous polyposis patients with APC gene mutations. Frontiers in Immunology, 14, 1163466.

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T Cell Phenotype Analysis After Infection

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Four days after infection, T cells were harvested and washed once with PBS, stained with rabbit anti-FLT3L antibody (Abcam, USA) for 1 h at 4 °C, and washed twice. Then PE donkey anti-rabbit IgG antibody (Biolegend, USA) was added, incubated at 4 °C for 30 min, and analyzed by flow cytometry using CantoII flow cytometer (BD Biosciences, San Jose, CA, USA) [14 (link)]. For T cell phenotype analysis, T cells were harvested 7 days after infection and washed once with PBS, stained with anti-CD4-PE/Cy7 (Biolegend, USA), anti-CD8-PerCP-Cy5.5 (Biolegend, USA), anti-CCR7-PE (Biolegend, USA), and anti-CD45RA-Pacific Blue (Biolegend USA) 30 min at 4 °C, then washed and resuspended in PBS for flow cytometry analysis [15 (link)].

Wang Y., Xu Y., Li S., Liu J., Xing Y., Xing H., Tian Z., Tang K., Rao Q., Wang M, & Wang J. (2018). Targeting FLT3 in acute myeloid leukemia using ligand-based chimeric antigen receptor-engineered T cells. Journal of Hematology & Oncology, 11, 60.

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T Cell Phenotyping by Flow Cytometry

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T cells were phenotyped by flow cytometry staining before (Day 0 – naïve and Day 10–10 days after 1^st round of stimulation) and after restimulation (Day 15), as well as in co-cultures with tumor cells. Briefly, CD8⁺ T cells were stained with LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit (Thermo Scientific), followed by surface staining at 4 ⁰C with: – anti-CD8 AF647, anti-CD8 PerCPCy5.5, anti-CD45RO Alexa Fluor 488, anti-CD45RA BV605, anti-CCR7-PE, anti-PD-1 PerCpCy5.5 (all from Biolegend) antibodies. Staining of CCR7 was performed at 37 ⁰C for 30 min. Intracellular granzyme B staining was performed with anti-Granzyme B-AF647 antibody (Biolegend), after permeabilization with Fixation/Permeabilization Solution Kit (BD Biosciences). Flow cytometric analysis was performed using a BD Fortessa. Data were analyzed using FlowJo (Tree Star).

Nelson N., Lopez-Pelaez M., Palazon A., Poon E., De La Roche M., Barry S., Valge-Archer V., Wilkinson R.W., Dovedi S.J, & Smith P.D. (2019). A cell-engineered system to assess tumor cell sensitivity to CD8+ T cell-mediated cytotoxicity. Oncoimmunology, 8(8), 1599635.

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Comprehensive Immunophenotyping by Flow Cytometry

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The following monoclonal antibodies were used for flow cytometry analysis and cell sorting; anti-CD8a-Alexa Fluor 488 (300916, BioLegend, 1:200), anti-CD8a-FITC (FITC-65135, Proteintech, 1:50), anti-CD3-PE (317307, BioLegend, 1:200), anti-CD4-APC (300552, BioLegend, 1:200), anti-CD45RA-FITC (304148, BioLegend, 1:200), anti-CD45RO-PerCP/Cy5.5 (304222, BioLegend, 1:200), anti-PD-1-APC/Cy7 (329921, BioLegend, 1:50), anti-CD57-PerCP/Cy5.5 (359621, BioLegend, 1:200), anti-IFNγ-APC (502511, Biolegend, 1:200), anti-TNFα-BV421 (502931, BioLegend, 1:100), anti-CCR7-PE (353203, BioLegend, 1:50), anti-TIGIT-PerCP/Cy5.5 (372717, BioLegend, 1:50), anti-pan HLA (M0736, Dako, 1:200), control mouse IgG2a (401501, BioLegend, 1:1111), and anti-mouse IgG-FITC (406001, BioLegend, 1:200). LIVE/DEAD Fixable-Near IR Dead Cell Stain Kit (L10119, Thermo Fisher Scientific, 1:400), LIVE/DEAD Fixable-Aqua Dead Cell Stain Kit (L34957, Thermo Fisher Scientific, 1:400), and Human BD Fc Block (564220, BD Pharmingen, 1:50) was also used. MHC tetramer was prepared using QuickSwitch Quant HLA-A^*24:02 Tetramer Kit-PE (TB-7302-K1, MBL Life Science) and QuickSwitch Quant HLA-A^*24:02 Tetramer Kit-BV421 (TB-7302-K4, MBL Life Science) with appropriate peptides. CMV pp65_341-349 peptide (TS-0020-P, MBL) was purchased and all the other peptides were synthesized with a purity of >95% (Scrum Inc., or Toyobo).

Ogura H., Gohda J., Lu X., Yamamoto M., Takesue Y., Son A., Doi S., Matsushita K., Isobe F., Fukuda Y., Huang T.P., Ueno T., Mambo N., Murakami H., Kawaguchi Y., Inoue J.I., Shirai K., Yamasaki S., Hirata J.I, & Ishido S. (2022). Dysfunctional Sars-CoV-2-M protein-specific cytotoxic T lymphocytes in patients recovering from severe COVID-19. Nature Communications, 13, 7063.

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