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Pd1 pe cy7

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PD1-PE/Cy7 is a fluorescently-labeled antibody targeting the PD-1 (Programmed Cell Death-1) cell surface receptor. It can be used for the detection and analysis of PD-1 expressing cells by flow cytometry.

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Lab products found in correlation

Cd3 fitc, by BioLegend (4 mentions) Cd4 bv711, by BioLegend (4 mentions) Facscanto 2, by BD (4 mentions) Zombie aqua, by BioLegend (3 mentions) Cd3 apc cy7, by BioLegend (3 mentions) Foxp3 fixation permeabilization kit, by Thermo Fisher Scientific (3 mentions) Pd l1 apc, by BioLegend (3 mentions) Live dead blue viability dye, by Thermo Fisher Scientific (3 mentions) Cd8α bv786, by BioLegend (3 mentions) Lag 3 percp cy5, by BioLegend (3 mentions) Ifn γ fitc, by BioLegend (3 mentions) Ionomycin, by Merck Group (3 mentions) Tigit pe cy7, by BioLegend (3 mentions) Icos apc, by BioLegend (2 mentions) Cd4 apc cy7, by BioLegend (2 mentions) Cd19 pe cy7, by BioLegend (2 mentions) Cd11c apc, by BioLegend (2 mentions) Cd56 fitc viobright, by Miltenyi Biotec (2 mentions) Nkg2a apc, by Miltenyi Biotec (2 mentions) Nkp30 bv421, by BioLegend (2 mentions)

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Anti-mouse PD1 PE-Cy7 (2 citations)

29 protocols using pd1 pe cy7

1

Multiparameter Flow Cytometry Immunophenotyping

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Unspecific binding was blocked by incubating cells with Fc receptor-blocking monoclonal antibody (clone 2.4G2; BD Biosciences) for 10 min. Cells were then stained with the following antibodies specifically binding: CD3-PerCP/Cy5.5 (145-2C11; BioLegend), CD4-BV785 (RM4-5; BioLegend), CD8-BV421 (53-6.7; BioLegend), B220/CD45R-APC-Cy7 (RA3-6B2; BioLegend), CD138-BV605 (281-2; BioLegend), CD69-PE (H1.2 F3; BioLegend), and PD-1-PE-Cy7 (29 F.1A12; BioLegend). Dead cells were excluded by ZombieGreen™ (BioLegend) staining, and doublets by scatter analysis. Cells were measured on a LSRII flow cytometer (BD) and analyzed by FlowJo software. In most samples, a minimum of 1 × 105 events were measured.
Keil A., Hall S.R., Körner M., Herrmann M., Schmid R.A, & Frese S. (2016). Suppression of lupus nephritis and skin lesions in MRL/lpr mice by administration of the topoisomerase I inhibitor irinotecan. Arthritis Research & Therapy, 18, 243.
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2

Comprehensive B and T Cell Phenotyping

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B cells were purified from the patient’s peripheral blood by positive selection using CD20 magnetic beads (Miltenyi Biotec, Cambridge, MA). CD4+ T cells were isolated from the peripheral blood of research subjects using the EasySep human CD4+ T cell enrichment kit (STEMCELL Technologies, Cambridge, MA). The following antibodies were used for flow cytometric staining: CD19 APC-Cy7, CD27 PerCP-Cy5.5, CD10 PE-Cy7, CD21 V450, CD69 PE, CD86 APC, FAS Alexa 647, CD4 APC-Cy7, CD127 PerCP-Cy5.5, CD45RO Alexa 700, CXCR5 PerCP-Cy5.5, PD-1 PE-Cy7, ICOS APC (all from Biolegend, San Diego, CA), CD3 eFluor 605NC (from eBioscience, San Diego, CA), and CD21 BD Horizon V450 (BD). Intracellular staining for FOXP3 Alexa 488 (eBioscience) and T-bet PE was performed using the FOXP3/Transcription Factor Staining Buffer Set (eBioscience) in accordance with the manufacturer’s directions.
Minto H., Mensah K.A., Reynolds P.R., Meffre E., Rubtsova K, & Gelfand E.W. (2019). A Novel ATM Mutation Associated with Elevated Atypical Lymphocyte Populations, Hyper-IgM, and Cutaneous Granulomas. Clinical immunology (Orlando, Fla.), 200, 55-63.
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3

Comprehensive Immune Cell Profiling of Adipose and Liver Tissues

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Liver and epigonadal visceral white adipose tissues were prepared for flow cytometry after cardiac perfusion with 10 mL HBSS (5 mM HEPES, 0.5 mM EDTA), as described (27 ). Following mechanical disruption, tissue was Liberase (Roche)-digested for 30 minutes at 37°C (shaking at 130 RPM) and filtered. Cells were separated on a 40% iodixanol density gradient by centrifugation at 1038 g for 25 minutes (no brake). Cells were blocked with anti-CD16/32, stained with Zombie Aqua (Biolegend) and antibodies (Biolegend): F4/80-FITC (BM8), Ly6C-PerCP/Cy5.5 (HK1.4), CD19-PE/Cy7 (6D5), CD11c-APC (N418), CD11b-AF700 (c1/70), I-A/I-E-APC/Cy7 (M5/114.15.2), CD45-PacBlue (30-F11), Ly6G-BV605 (1A8), CD3e-FITC (145–2C11), CD62L-PE-dazzle (MEL-14), CD8a-PerCP/Cy5.5 (53–6.7), PD-1-PE/Cy7 (29F.1A12), CD4-APC (GK1.5), CD44-BV605 (IM7). Beckman Coulter CytoFLEX and FlowJo were used to assess immune cell populations using the following gating strategy: Cells, singlets, live cells, CD4+ T cells (CD45+CD3e+CD4+), CD8+ T cells (CD45+CD3e+CD8+), B cells (CD45+CD19+), Kupffer cells (liver only, CD45+F480hiCD11blo), macrophages (CD45+F480intCD11bhiLy6clo), inflammatory monocytes (CD45+F480intCD11bhiLy6chi), dendritic cells (CD45+F480−CD11c+MHCII+), and neutrophils (CD45+F480−CD11b+ Ly6G+SSChi).
Melchor S.J., Saunders C.M., Sanders I., Hatter J.A., Byrnes K.A., Coutermarsh-Ott S, & Ewald S.E. (2020). IL-1R regulates disease tolerance and cachexia in T. gondii infection. Journal of immunology (Baltimore, Md. : 1950), 204(12), 3329-3338.
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4

Quantifying NK Cell Markers in Whole Blood and Tumors

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Whole blood and intratumoural immune cells were stained with CD56-FITC-Viobright, NKG2A-APC, CCR5-FITC, CCR1-APC, CCR3-PE (Miltenyi Biotec), CD3-APC-Cy7, NKp30-BV421, NKp46-PE-Cy7, NKG2D-PE-Cy5, PD-1-PE-Cy7, TIGIT-PE-Cy5, CD69-BV510 (BioLegend). Red blood cells were lysed using BD Lysing Solution (BD Biosciences) as per manufacturer’s instructions. NK cells were quantified as CD56+CD3− cells within the lymphocyte gate. Cells were acquired using the CANTO II (BD Biosciences) flow cytometer and analysed using FlowJo software (Tree Star).
Mylod E., O’Connell F., Donlon N.E., Davern M., Marion C., Butler C., Reynolds J.V., Lysaght J, & Conroy M.J. (2024). Real-time ex vivo monitoring of NK cell migration toward obesity-associated oesophageal adenocarcinoma following modulation of CX3CR1. Scientific Reports, 14, 4017.
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5

Flow Cytometric Analysis of T Cell Subsets

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Spleen cells were obtained from non-immunized mice, self-cured mice 25 days post P. yoelii 17X infection and immunized mice with rexPy or rexC on day 20 after the second immunization, as described above. Splenocytes were analyzed for the expression of different markers with an LSRFortessa flow cytometer and data were analyzed with FlowJo software. T cells subsets were identified according to lymphocytes' SSC-A/FSC-A profile and labeling with CD4-PerCp or CD8-Alexa Fluor-700 antibodies (Biolegend). The phenotype of CD4+ and CD8+T cells in spleen was analyzed with a panel of fluorochrome-conjugated antibodies obtained from Biolegend that included: CD62L-Pacific Blue, PD1-PE/Cy7, CD127-PE and CD44-FITC.
Martín-Jaular L., de Menezes-Neto A., Monguió-Tortajada M., Elizalde-Torrent A., Díaz-Varela M., Fernández-Becerra C., Borras F.E., Montoya M, & del Portillo H.A. (2016). Spleen-Dependent Immune Protection Elicited by CpG Adjuvanted Reticulocyte-Derived Exosomes from Malaria Infection Is Associated with Changes in T cell Subsets' Distribution. Frontiers in Cell and Developmental Biology, 4, 131.
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6

Comprehensive Immune Phenotyping Protocol

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The following anti-human antibodies were used for staining: CD3-FITC, CD3- Alexa Fluor 700, CD8a-Alexa Fluor700, CD4-Percp-Cy5.5, CD4-Alexa Fluor 700, CD25-PE, CD25-PE-Cy7, Foxp3-FITC, Foxp3- Percp-Cy5.5, Foxp3-BV421, CD127-APC, CD39-APC, LAP-PE, IL-10-PE, TIM-3-PE, TIM-3-BV421, IFN-γ receptor-PE, IFN-γ- APC, Granzyme B-PE-dazzle E, PD-1-APC, PD-1- PE-Cy7, PD-1- Percp-Cy5.5, CTLA-4-PE, CTLA-4-FITC, ki67-Alexa Fluor 488 and their respective isotype controls were purchased from Biolegend. Recombinant human IFN-γ (R&D Systems) was used at 200 ng/ml. Anti-PD-1 Ab (Nivolumab from Bristol-Myers Squibb), anti-Tim-3 (clone 2E2 from Biolegend) and isotype were used at 10µg/ml.
Liu Z., McMichael E.L., Shayan G., Li J., Chen K., Srivastava R., Kane L.P., Lu B, & Ferris R.L. (2018). Novel effector phenotype of Tim-3+ regulatory T cells leads to enhanced suppressive function in head and neck cancer patients. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(18), 4529-4538.
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7

Multiparametric T cell analysis

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T cells were re-stimulated with phorbol myristate acetate (Sigma, 50 ng/mL) and ionomycin (Sigma, 500 ng/mL). 90 minutes later, cells were treated with Brefeldin A to block cytokine secretion. 3 h later, cells were stained for surface markers and simultaneously labelled with Live/Dead Blue Viability Dye (Thermo Fisher) for 20 minutes at 4°C. Cells were washed twice and fixed overnight using a FoxP3 Fixation/Permeabilization kit (Thermo Fisher). The following day, cells were washed and stained for intracellular cytokines at room temperature for 1 h. They were then washed 3 times and analysed using an LSR Fortessa machine (Beckman Dickinson). Analysis of mean fluorescence intensity was performed using FlowJo v10.0. All experiments were performed at least two independent times. Antibodies used (at 1:100 unless otherwise noted) were TNF-PE (BioLegend, MP6-XT22, 506306), PD-1-PECy7 (BioLegend, RMP1–30, 109110) IFN-γ-FITC (BioLegend, XMG1.2, 505806), CD4-BV711 (BioLegend, RM4–5, 100550), CD8α-BV786 (BioLegend, 53–6.7, 100750), Tox-PE (Miltenyi, REA473, 130–120-716, 1:50), Tcf7-Alexa647 (Cell Signaling, C63D9, 6709), PD-L1-APC (BioLegend, 10F.9G2, 124312, 1:5000), LAG-3-PerCP-Cy5.5 (BioLegend, C9B7W, 125211), TIM-3-PE-Dazzle594 (BioLegend, B8.2C12, 134013).
Vardhana S.A., Hwee M.A., Berisa M., Wells D.K., Yost K.E., King B., Smith M., Herrera P.S., Chang H.Y., Satpathy A.T., van den Brink M.R., Cross J.R, & Thompson C.B. (2020). Impaired mitochondrial oxidative phosphorylation limits the self-renewal of T cells exposed to persistent antigen. Nature immunology, 21(9), 1022-1033.
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8

Comprehensive Immune Cell Profiling

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Vehicle-, nivolumab- or ipilimumab-expanded lymphocytes were stained with zombie NIR viability dye, CD8-BV421, CD4-BV510, CD3-FITC, CXCR3-PE, CCR6-APC, CTLA-4-PerCPCy5.5, TIGIT-PE/Cy7, PD-L1-PerCPCy5.5, PD-1-PECy/7, CD25-FITC and FOXP3-BV421 (Biolegend, USA). Intracellular FoxP3 permeabilization and fixation buffer was used for FoxP3 staining (Biolegend, USA). Cells were acquired using the BD FACs Canto II (BD Biosciences) using Diva software and analysed using FlowJoTM v10.7.
Davern M., Gaughan C., O’ Connell F., Moran B., Mylod E., Sheppard A.D., Ramjit S., Yun-Tong Kung J., Phelan J.J., Davey M.G., Ryan E.J., Butler C., Quinn L., Howard C., Tone E., Phoenix E., Butt W.T., Lynam-Lennon N., Maher S.G., Ravi N., Donohoe C.L., Reynolds J.V., Lysaght J, & Donlon N.E. (2023). PD-1 blockade attenuates surgery-mediated immunosuppression and boosts Th1 immunity perioperatively in oesophagogastric junctional adenocarcinoma. Frontiers in Immunology, 14, 1150754.
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9

Multiparametric Flow Cytometry Staining

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Staining for surface markers was performed by incubating cells with antibody at 1:200 dilutions in fluorescence-activated cell sorting buffer (0.1% BSA in PBS) for 30 min at 4 °C. For intracellular cytokine staining of IFNγ and TNFα, surface markers were stained before fixation/permeabilization (BD Cytofix/Cytoperm Kit, #554714; BD Biosciences) (40 (link)). Flow antibodies were purchased from BioLegend with the information as follows: CD8a-BV786 (#563332), CD11c-BV786 (#563735), PD1-PE/Cy7 (#109109), TIM3-APC (#119705), KLRG1-FITC (#138409), LAG3-PerCP/Cy5.5 (#125211), TNFα-PE/Cy7 (#506323), and Granzyme B-Alexa 700 (#372221). Samples were acquired on BD LSRFortessa flow cytometer and analyzed with FlowJo software (https://www.flowjo.com/solutions/flowjo) (Tree Star) unless otherwise stated.
Zhang J., Ye Z.W., Bräutigam L., Chakraborty P., Luo Z., Culpepper J., Aslam M., Zhang L., Johansson K., Haeggström J.Z., Xu J., Olsson M., Townsend D.M., Mehrotra S., Morgenstern R, & Tew K.D. (2023). A role for microsomal glutathione transferase 1 in melanin biosynthesis and melanoma progression. The Journal of Biological Chemistry, 299(8), 104920.
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10

Comprehensive Immune Cell Profiling

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The following anti-human mAbs were used for staining: HLA-DR-APC, CD11b-PErCP-Cy5.5, CD163-BV421, CD33-BV421, CD8-APC, PD-1-PE-Cy7, Tim-3-BV421, TIGIT-APC, CD27-Alexa Fluor 700, LAP-BV421, FoxP3-PerCP-Cy5.5 and PD-L1-PE purchased from Biolegend (San Diego, CA); CD11b-PE, CD1c-APC-Cy7, CD141-BV711, CD4-PerCP-Cy5.5, CD56-Alexa Fluor 700, CD3-Alexa Fluor 700, CD19-Alexa Fluor 700, CD16-PE-Cy7 , HLA-A2-APC-H7, CD73-PE, purchased from BD Biosciences (San Jose, CA); CD14-PE-TR purchased from Life Technologies (Carlsbad, CA); CD86-FITC purchased from R&D Systems (Minneapolis, MN); CTLA-4-FITC purchased from Ancell (Bayport, MN); and the PE-labeled HLA-A*0201-EGFR853-861 tetramer obtained from the Tetramer Facility of the National Institute of Allergy and Infectious Disease (Atlanta, GA).
Intracellular staining of FoxP3 was performed as follows: PBL or TIL were stained with surface marker antibodies, fixed with fixation/permeabilization buffer (eBioscience), washed, and stained for intracellular antigens in 1X permeabilization buffer. Cells were analyzed on an LSR Fortessa (BD) flow cytometer, and data analyzed using Flow Jo (Treestar, Ashland, OR). Dead cells were excluded based on viability dye staining (Zombie Aqua Fixable Viability Dye, Biolegend).
Shayan G., Kansy B.A., Gibson S.P., Srivastava R.M., Bryan J.K., Bauman J.E., Ohr J., Kim S., Duvvuri U., Clump D.A., Heron D.E., Johnson J.T., Hershberg R.M, & Ferris R.L. (2017). Phase Ib Study of Immune Biomarker Modulation with Neoadjuvant Cetuximab and TLR8 stimulation in Head and Neck Cancer to Overcome Suppressive Myeloid Signals. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(1), 62-72.
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