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Ccr7 pe cf594

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CCR7-PE-CF594 is a fluorescently conjugated antibody used for the detection and analysis of CCR7 (C-C chemokine receptor type 7) expression on cells. It is designed for use in flow cytometry applications.

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Lab products found in correlation

Facsdiva software, by BD (2 mentions) Cd69 bv650, by BioLegend (2 mentions) Cd3 af700, by Thermo Fisher Scientific (2 mentions) Cd27 apc, by BD (2 mentions) Cd45ra fitc, by BD (2 mentions) Cd4 alexa fluor 700, by Thermo Fisher Scientific (2 mentions) Cd39 pe cy7, by Thermo Fisher Scientific (2 mentions) Cd8 apc, by Thermo Fisher Scientific (2 mentions) Ccr7 cd197 pe cy7, by Thermo Fisher Scientific (2 mentions) Pd1 cd279 pe, by Thermo Fisher Scientific (2 mentions) Cd73 fitc, by Thermo Fisher Scientific (2 mentions) Hla dr bv421, by BD (2 mentions) Cd25 bv421, by BD (2 mentions) Cd127 pe cf594, by BD (2 mentions) Cd103 bv421, by BD (2 mentions) Navios cytometer, by Beckman Coulter (2 mentions) Cd8 v500, by BD (1 mentions) Cd38 pe cy7, by BioLegend (1 mentions) Zombie fixable viability kit, by BioLegend (1 mentions) Cd4 af700, by BD (1 mentions)

11 protocols using ccr7 pe cf594

1

Comprehensive Immunophenotyping by Flow Cytometry

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All cells were stained for viability using the Zombie Fixable Viability Kit (Biolegend), incubated with anti-CD16/32 Fc-block (BioXcell), and stained with the indicated antibodies: CD19 APC, CD3 APC-H7 or BV450, CD4 AF700, CD8 V500, CCR7 PE-CF594, CD69 BV785, CCR5 PE, CD103 FITC, (BD Biosciences) CD38 PE-Cy7, CD11c BV711, CD14 BV650, CD45RA BV605 (Biolegend). Stained samples were run on an LSRII flow cytometer, data acquired using FACS DIVA software (BD Biosciences) and analyzed using FlowJo software (TreeStar, Inc., Ashland, Oregon).
Swaims-Kohlmeier A., Haaland R.E., Haddad L.B., Sheth A.N., Evans-Strickfaden T., Lupo L.D., Cordes S., Aguirre A.J., Lupoli K.A., Chen C.Y., Ofotukun I., Hart C.E, & Kohlmeier J.E. (2016). Progesterone levels associate with a novel population of CCR5+ CD38+ CD4 T cells resident in the genital mucosa with lymphoid trafficking potential. Journal of immunology (Baltimore, Md. : 1950), 197(1), 368-376.
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2

Multiparameter Flow Cytometry Phenotyping

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Standard extracellular staining (20 mins at 24°C) was performed using the following fluorophore-labeled antibodies: CD3-BUV737 (BD Biosciences), CD4-BUV395 (BD Biosciences), CD8-BV711 (BD Biosciences), CD14-BV510 (BD Biosciences), CD19-BV510 (BioLegend), Live/Dead Fixable Aqua (ThermoFisher), CCR7-PE-CF594 (BD Biosciences), CD45RA-APC-H7 (BD Biosciences), CD28-APC-R700 (BD Biosciences), CD57-PE (BioLegend), PD-1-BV605, BV421 (BioLegend), CD69-BV650 (BioLegend), CD38-BV421 (BioLegend), HLA-DR-PerCP-Cy5.5 (BD Biosciences), TIGIT-PE-Cy7 (BioLegend), DNAM-BB515 (BD Biosciences), FcγRIIB-BV786 (BD Biosciences). Fluorescence minus one (FMO) controls were used to determine negative expression of FcγRIIB. Apoptosis was measured with Caspase-3/7 Green Flow Cytometry Assay Kit (ThermoFisher) or FITC Annexin V with 7-AAD Viability Staining Solution (BioLegend), according to the manufacturer’s instructions.
Sun H., Hartigan C.R., Chen C.W., Sun Y., Tariq M., Robertson J.M., Krummey S.M., Mehta A.K, & Ford M.L. (2021). TIGIT regulates apoptosis of risky memory T cell subsets implicated in belatacept-resistant rejection. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 21(10), 3256-3267.
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3

Flow Cytometry of Immune Cell Subsets

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The following fluorochrome conjugated antihuman monoclonal antibodies (MoAbs) were used for flow cytometry studies: ICOSBV421, ICOSAlexa488, CD40LBV605, CD69BV650, HLA-DRFITC, CD38APCCy7, and TNF-αAPCCy7 from BioLegend (San Diego, CA); CD3BUV395, CD4PerCPCy5.5, CD8Alexa-Fluor700, CCR7PECF594, IL-2BV711, CXCR5Alexa647, IFNγPE-Cy7, PD1BV650, CD45ROAPCH7, CD21PECy5, CD27PerCPCy5.5, IgDFITC, CD10PECy7, and CD20Alexa700 from BD Bioscience (San Jose, CA); IL-21PE, CD27PECy5, and IL-17Alexa488 from e-Biosciences (San Diego, CA); and CD45ROPE-TexasRed from Beckman Coulter (Fullerton, CA). Live/Dead Fixable Aqua Dead Cell Stain Kit and CellTrace Violet Cell Proliferation Kit were from ThermoFisher (Boston, MA). Recombinant human IL-21 (Cat #8879-IL-010) from R&D systems, purified antihuman IL-2 (Cat #3440-ON-500) and antihuman-IL-21 (Cat #MT216G/21.3m) from Mabtech, and antihumanTNF-α (Cat #502922) from BioLegend were used. Samples were acquired on a BD LSRFortessa (BD Biosciences, CA) flow cytometer and analyzed by FlowJo V10 (TreeStar, Inc).
Pallikkuth S., de Armas L.R., Rinaldi S., George V.K., Pan L., Arheart K.L., Pahwa R, & Pahwa S. (2019). Dysfunctional peripheral T follicular helper cells dominate in people with impaired influenza vaccine responses: Results from the FLORAH study. PLoS Biology, 17(5), e3000257.
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4

Phenotypic Characterization of NY-ESO-1 TCR-Transduced T Cells

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Frozen patient PBMCs and healthy donor NY-ESO-1 TCR transduced T cells were thawed in RPMI with 10% FBS. These cells were then stained with a pentamer specific to NY-ESO-1c259TCR-transduced T cells (A*02:01 SLLMWITQC) for 20 minutes at 4°C, followed by subsequent staining with antibodies for an additional 15 minutes at 4°C. 7-AAD was used as the live/dead marker and was added at least 10 minutes prior to sorting. Fluorescence minus one controls were prepared and used for correct gate compensation settings. Cells were sorted using an Aria Fusion cell sorter (BD Biosciences). Data were analyzed using FACSDiva (BD Biosciences) software postsort. The following antibodies were used for identification of transduced T cells for sorting and subsequent postsort phenotypic analysis: CD8-QDOT655 (Life Technologies), CD4-AF780 (eBioscience), CD3-AF700 (eBioscience), CCR7-PE-CF594 (BD Biosciences) and CD45RA-FITC (eBioscience), CD127-BV421 (BioLegend), CD95-BV711 (BD Biosciences). The NY-ESO-1 Pro5 MHC-pentamer conjugated to PE (A*02:01 SLLMWITQC) was purchased from ProImmune. For lineage-tracing studies, NY-ESO-1 TCR-transduced T cells were stained with NY-ESO-1 dextramer conjugated to PE (A*02:01 SLLMWITQC) and purchased from Immudex. 7-AAD was purchased from BD Biosciences.
D'Angelo S.P., Melchiori L., Merchant M.S., Bernstein D., Glod J., Kaplan R., Grupp S., Tap W.D., Chagin K., Binder G.K., Basu S., Lowther D.E., Wang R., Bath N., Tipping A., Betts G., Ramachandran I., Navenot J.M., Zhang H., Wells D.K., Winkle E.V., Kari G., Trivedi T., Holdich T., Pandite L., Amado R, & Mackall C.L. (2018). Antitumor Activity Associated with Prolonged Persistence of Adoptively Transferred NY-ESO-1c259T Cells in Synovial Sarcoma. Cancer discovery, 8(8), 944-957.
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5

Comprehensive PBMC Functional Profiling

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Thawed PBMC were rested for 12 hours prior to stimulation of 1.5 million cells each with Brefeldin A (Sigma Aldrich) and either gag peptides (1 ug/peptide/mL; NIH AIDS reagent program); Staphylococcal enterotoxin B (SEB; 1 ng/mL) or Dimethyl sufoxide (DMSO) for 6 hours. Cells were then surface stained with CD3 AlexaFluor700, CD8 Pacific Blue, CCR7 PE-CF594, PD-1 PE-Cy7 (all BD Biosciences), CD4 Qdot 605 (Invitrogen), CD45RA Brilliant Violet 650, CD19 Brilliant Violet 510 (Biolegend), CD27 APCe780, and aqua fluorescent reactive dye (Invitrogen), permeabilised with Saponin and stained intracellularly with IL-2 PerCP-Cy5.5, IFNγ APC and TNFα Alexa Fluor 488 (all BD Biosciences) prior to fixation. Cells were acquired within 24 hrs using a BD LSR-II and analysed using FlowJo version 9 and 10.
Elliott J.H., Wightman F., Solomon A., Ghneim K., Ahlers J., Cameron M.J., Smith M.Z., Spelman T., McMahon J., Velayudham P., Brown G., Roney J., Watson J., Prince M.H., Hoy J.F., Chomont N., Fromentin R., Procopio F.A., Zeidan J., Palmer S., Odevall L., Johnstone R.W., Martin B.P., Sinclair E., Deeks S.G., Hazuda D.J., Cameron P.U., Sékaly R.P, & Lewin S.R. (2014). Activation of HIV Transcription with Short-Course Vorinostat in HIV-Infected Patients on Suppressive Antiretroviral Therapy. PLoS Pathogens, 10(11), e1004473.
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6

Comprehensive T Cell Subset Analysis

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For T cell subset analyses, 100 μL of whole blood was mixed with an optimal dilution of the following monoclonal antibodies: CD3-APC-CY7, CD4-PE, CD8-PerCP-Cy5.5, CD45RA-FITC, CCR7-PE-CF594, CD5-PE-CY7, CD27-APC, CD127-V450, CD25-BV510 (BD Bioscience). After adding all antibodies, the mixture was gently vortexed and incubated for 15 min in the dark at room temperature. Subsequently, 2 mL of 1× FACS lysing solution (BD Bioscience) was added and incubated for 10 min in the dark at room temperature. Finally, cells were washed 3 times with 200 μL washing buffer. Data was collected by a 9-colour Aria I flow cytometer (BD Bioscience) and analyzed with FACSDiva software (version 6.1.3, BD Bioscience). The following T cell subsets were determined (Mahnke et al., 2012; Schatorje et al., 2012)
Tem), and CD8 + transitional memory T cells (CD3 + CD8 + CD4 -CD45RA -CCR7 -CD27 + , CD8 + Ttm). The full-minus-one (FMO) principle was adopted for gating the T cell subsets. Absolute counts of each subgroup were calculated by multiplying the total lymphocyte counts with the percentages of each subset that were determined by flow cytometry.
Cao J., Xu X., Zhang Y., Zeng Z., Hylkema M.N, & Huo X. (2018). Increased memory T cell populations in Pb-exposed children from an e-waste-recycling area. The Science of the total environment, 616-617.
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7

Multiparameter Flow Cytometry for Detecting Antigen-Specific T Cells

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The following antibodies were used: cluster of differentiation (CD)4 BV605, CD8 BV650, CCR7 PE-CF594 (Becton Dickinson), CD45RA PECy7, CD28 BV421, CD127 BV711, PD-1 PerCP-Cy5.5, CD95 APC, CD62L BV785 (BioLegend, Dedham, MA), CD3 AF700, CD45RO FITC, CD27 APC-ef780 (eBioscience, San Diego, CA). The dead cell exclusion stain (Live/Dead Aqua) was purchased from Invitrogen (Carlsbad, CA). To detect NY-ESO-1c259TCR-expressing cells, purified anti-phycoerythrin-conjugated dextramer reagents specific for the HLA-A*02:01 SLLMWITQC complex (Immudex) were used at the manufacturer’s recommended concentrations.
Ramachandran I., Lowther D.E., Dryer-Minnerly R., Wang R., Fayngerts S., Nunez D., Betts G., Bath N., Tipping A.J., Melchiori L., Navenot J.M., Glod J., Mackall C.L., D’Angelo S.P., Araujo D.M., Chow W.A., Demetri G.D., Druta M., Van Tine B.A., Grupp S.A., Abdul Razak A.R., Wilky B., Iyengar M., Trivedi T., Winkle E.V., Chagin K., Amado R., Binder G.K, & Basu S. (2019). Systemic and local immunity following adoptive transfer of NY-ESO-1 SPEAR T cells in synovial sarcoma. Journal for Immunotherapy of Cancer, 7, 276.
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8

Phenotypic Characterization of Regulatory T Cells

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The following anti-human monoclonal antibodies (mAbs) were used for flow cytometry: CD4 Alexa Fluor® 700, CD8 APC, CD39 PE-Cy7, CD73 FITC, and CD73 eFluor450, CCR7 (CD197) PE-Cy7, PD1 (CD279) PE (eBioscience); CCR7 PE-CF594, CD45 AmCyan and CD45 FITC (Becton Dickinson), CD3 APC-H7, CD25 FITC (BioLegend) and CD25 PE (MACS Miltenyi). All mAbs were titrated using normal PBL to establish optimal dilution. Treg were defined by their ability to produce extracellular adenosine as previously described by Deaglio and Borselino [28 (link), 29 (link)]. We have previously demonstrated that these CD4+ CD39+ CD25+ Treg are FOXP3+ CD127neg [24 (link)].
Jeske S.S., Schuler P.J., Doescher J., Theodoraki M.N., Laban S., Brunner C., Hoffmann T.K, & Wigand M.C. (2020). Age-related changes in T lymphocytes of patients with head and neck squamous cell carcinoma. Immunity & Ageing : I & A, 17, 3.
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9

Multiparametric Flow Cytometry Panel

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The following anti-human monoclonal antibodies (mAbs) were used for flow cytometry: CD4 Alexa Fluor ® 700, CD8 APC, CD39 PE-Cy7, CD73 FITC, and CD73 eFluor450, CCR7 (CD197) PE-Cy7, PD-1 (CD279) PE (eBioscience); CD19 PE-Cy5, CCR7 PE-CF594, CD45 AmCyan and CD45 FITC (Becton Dickinson), CD3 APC-H7, CD25 FITC, CD25 PE (MACS Miltenyi). All mAbs were titrated using PBMC of healthy donors to establish optimal dilution.
Jeske S.S., Weissinger S.E., Veit J.A., Brunner C., Huber U., Theodoraki M.N., Hoffmann T.K., Schuler P.J, & Doescher J. (2019). Treatment-induced changes of lymphocyte subsets in patients with adenoid cystic carcinoma of the head and neck. European archives of oto-rhino-laryngology : official journal of the European Federation of Oto-Rhino-Laryngological Societies (EUFOS) : affiliated with the German Society for Oto-Rhino-Laryngology - Head and Neck Surgery, 276(5).
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10

SARS-CoV-2-Specific T Cell Activation

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We analyzed the cell composition of the T cells specifically activated by SARS-CoV-2 in the IFN-γ assay by subtracting the basal cytokine response from the background control. We stained the cell surface for 20 min at 4°C using the following fluorochrome-conjugated antibodies titrated to their optimal concentrations: CD45RA FITC (BD Pharmingen), CD27 APC (BD Pharmingen), CD3 VioGreen (Miltenyi Biotec), CD4 PECy7 (BD Pharmingen), CD8 APC Cy7 (BD Pharmingen), and 7AAD (BD Horizon). For the Treg panel CD25 BV421 (BD Horizon) and CD127 PE-CF594 (BD Horizon), were used. For the activation panel, HLA-DR BV 421 (BD Pharmingen), CD69 BV421 (Biolegend), and CD25 BV421 (BD Horizon), were used. For the exhaustion panel PD1 AF700 (Biolegend) and NKG2A BV421 (Biolegend) were used. For the chemokine panel, CD103 BV421 (BD Horizon) and CCR7 PE-CF594 (BD Horizon) were used. Cell acquisition was performed using a Navios cytometer (Beckman Coulter), acquiring an average of 200,000 cells. The analysis was performed using FlowJo 10.7.1 (FlowJo LLC).
Ferreras C., Pascual-Miguel B., Mestre-Durán C., Navarro-Zapata A., Clares-Villa L., Martín-Cortázar C., De Paz R., Marcos A., Vicario J.L., Balas A., García-Sánchez F., Eguizabal C., Solano C., Mora-Rillo M., Soria B, & Pérez-Martínez A. (2021). SARS-CoV-2-Specific Memory T Lymphocytes From COVID-19 Convalescent Donors: Identification, Biobanking, and Large-Scale Production for Adoptive Cell Therapy. Frontiers in Cell and Developmental Biology, 9, 620730.
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