Pd 1 percp cy5

Manufactured by BioLegend

Sourced in United States

PD-1-PerCP-Cy5.5 is a fluorescently-labeled antibody that specifically binds to the programmed cell death-1 (PD-1) receptor. It is designed for the detection and analysis of PD-1-expressing cells using flow cytometry.

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Close spelling variants of this product

Anti-PD-1 PerCP-Cy5.5 (EH12.2H7, PerCP/Cy5.5 anti-human CD279/PD-1 PD-1-PerCP/Cy5.5 (EH12.2H7, PerCP/Cy5.5-PD-1 PerCP-Cy5.5

10 protocols using pd 1 percp cy5

Quantification of Immune Cell Subsets

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Counting of Th17, Treg, exhausted T, and exhausted Treg cell was performed using flow cytometry. The monoclonal antibodies of surface and intracellular antigens used for counting Th17 cells were CD4-FITC (BioLegend, US, cat. no. 357405) and IL-17A-PE (BioLegend, US, cat. no. 506903), and CD4-FITC (BioLegend, US, cat. no. 357405), CD25-PE (BioLegend, US, cat. no. 985802), and CD127-PerCP-Cy5.5 (BioLegend, US, cat. no. 351321) were used for Treg cells; CD8-FITC (BioLegend, US, cat. no. 980908), PD-1-PerCP-Cy5.5 (BioLegend, US, cat. no. 329913), and Tim-3-PE (BioLegend, US, cat. no. 345006) for exhausted T cells; and CD4-FITC (BioLegend, US, cat. no. 357405), CD25-PE (BioLegend, US, cat. no. 985802), and PD1-PerCP-Cy5.5 (BioLegend, US, cat. no. 329913) for exhausted Treg cells [18 (link)].

Yan Y.N., Zhang J., Yang N., Chen C, & Li W. (2023). T Cell Subsets and the Expression of Related MicroRNAs in Patients with Recurrent Early Pregnancy Loss. Mediators of Inflammation, 2023, 8215567.

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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-1^c259TCR-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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Multiparametric Flow Cytometry Analysis

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Single-cell suspensions from spleens, lymph nodes, and thymi were incubated with anti-CD16/32 at 5 µg/ml for 20 min on ice to block Fc receptors. Cells were stained with the following antibodies: CD4 PerCP-Cy55, TCRβ PerCp-Cy55, and CD62L APC (TONBO); CD44 FITC, PD-1 PerCP-Cy55, CD69 Cy7PE, CD25 PerCP-Cy55 (Biolegend); CD4 BUV395, and CD8 BUV737 (BD); Vα2 PE (phycoerythrin), Vα2 Pacific Blue, Vα2 FITC, FR4 Cy7PE, and CD73 BV605 (eBioscience); and CD25 BV605 (Life Technology). Dead cells were excluded using the live/dead fixable Near-IR death cell stain kit (Invitrogen). Intracellular Foxp3-FITC staining was done according to the manufacturer’s instructions (Life Technology). For detection of negatively selected thymocytes, caspase 3 PE (BD) was used as previously described (Breed et al., 2019 (link)). For intracellular flow cytometry, antibodies against phosphorylated ERK^T202/Y204 (mAb 197G2), AKT^S473 (Cell Signaling) were used as previously described (Hsu et al., 2009 (link)). Antibody against Alexa 647–anti-rabbit IgG was used as a secondary antibody to detect phosphorylation of ERK and AKT.

Nguyen T.T., Wang Z.E., Shen L., Schroeder A., Eckalbar W, & Weiss A. (2021). Cbl-b deficiency prevents functional but not phenotypic T cell anergy. The Journal of Experimental Medicine, 218(7), e20202477.

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T Cell Phenotyping and Intracellular Staining

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PBMCs were acquired, and fluorescence antibodies against human CD4-APC-Cy7 (Biolegend, isotype: mouse IgG2b, κ, clone: OK74, cat: 317418) or CD4-PE (Biolegend, isotype: mouse IgG2b, κ, clone: OKT4, cat: 317410), CXCR5-PECy7 (Biolegend, isotype: mouse IgG1, κ, clone: J252D4, cat:356924), PD-1-PerCP-Cy5.5 (Biolegend, isotype: mouse IgG2b, κ, clone: A17188B, cat: 621613), and ICOS-APC-Cy7 (Biolegend, isotype: Armenian Hamster IgG, clone: C398.4A, cat: 313529) were used for cell surface staining. After half an hour, the cells were washed twice with PBS. A transcription factor staining buffer kit (TONBO, San Diego, CA) was used for intracellular staining. Fluorescence antibodies against FOXP3-APC (Invitrogen, isotype: rat IgG2a, κ, clone: PCH101, cat: 17-4776-42), Helios-FITC (Biolegend, isotype: Armenian Hamster IgG, clone: 22F6, cat: 137214), IL-10-PerCP-Cy5.5 (Biolegend, isotype: rat IgG1, κ, clone: JES3-907, cat:501418) and IL-21-PE (Invitrogen, isotype: mouse IgG1, κ, clone: 3A3-N2, cat: 12-7219-41) were used for intracellular staining. Then, the cells were suspended in PBS and analyzed by a FACSCanto cytometer (BD Biosciences, San Jose, CA, USA).

Zeng X., Alimu X., Bahabayi A., Zhang Z., Zheng M., Yuan Z., Liu T, & Liu C. (2024). Helios characterized circulating follicular helper T cells with enhanced functional phenotypes and was increased in patients with systemic lupus erythematosus. Clinical and Experimental Medicine, 24(1), 5.

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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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Multiparametric Flow Cytometry Immunophenotyping

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The following anti-human monoclonal antibodies (mAb) were used for staining: PD-1-APC (eBioscience, San Diego, CA, USA), CD3-PerCP-Cy5.5, CTLA-4-PE, PD-1-PerCP Cy5.5 (all Biolegend, San Diego, CA, USA), CCR7-FITC (R&D Systems Inc, Minneapolis, MN, USA) CD3-Alexa Fluor 405, CD4-PE-Texas Red, CD-8-PE-TR, CD45RA-PE-TR (all Life Technologies, Grand Island, NY, USA), CD3-APC-Cy7, CD8-APC, CD8-PE-Cy7 (all BD Biosciences, San Jose, CA, USA), granzyme B FITC (clone GB11, Biolegend) including their respective isotypes, which served as negative controls for surface as well as intracellular staining. Antibodies were pre-titrated using activated as well as non-activated PBMC to determine optimal staining dilutions. Viability was assessed by Zombie aqua (Biolegend)

Kansy B.A., Concha-Benavente F., Srivastava R.M., Jie H.B., Shayan G., Lei Y., Moskovitz J., Moy J., Li J., Brandau S., Lang S., Schmitt N.C., Freeman G.J., Gooding W.E., Clump D.A, & Ferris R.L. (2017). PD-1 status in CD8+ T cells associates with survival and anti-PD-1 therapeutic outcomes in head and neck cancer. Cancer research, 77(22), 6353-6364.

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Expanded TIL Immunophenotyping by Flow Cytometry

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Expanded TIL were stained in 100 µL of FACS Wash Buffer (Dulbecco’s Phosphate Buffered Saline 1× with 1% Bovine Serum Albumin) for 30 min using fluorochrome-conjugated monoclonal antibodies for CD3 (FITC), CD16 (PE), γδTCR (APC), CD56 (PE-Cy7), CD4 (PerCP Cy5.5), CD8 (PB), BLTA (PE), CD28 (PE-Cy7), TIM3 (APC) (all BD Bioscience, San Jose, CA), PD-1 (PerCP-Cy5.5) (Biolegend, San Diego, CA) and AQUA live/dead dye (Invitrogen, Carlsbad, CA). Stained cells were acquired using the BD FACScanto II and analyzed using FlowJo software v 10.1 (Tree star, Ashland, OR).

Tavera R.J., Forget M.A., Kim Y.U., Sakellariou-Thompson D., Creasy C.A., Bhatta A., Fulbright O.J., Ramachandran R., Thorsen S.T., Flores E., Wahl A., Gonzalez A.M., Toth C., Wardell S., Mansaray R., Radvanyi L.G., Gombos D.S., Patel S.P., Hwu P., Amaria R.N., Bernatchez C, & Haymaker C. (2018). Utilizing T-cell activation signals 1, 2 and 3 for tumor-infiltrating lymphocytes (TIL) expansion: the advantage over the sole use of interleukin-2 in cutaneous and uveal melanoma. Journal of immunotherapy (Hagerstown, Md. : 1997), 41(9), 399-405.

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Flow Cytometric Analysis of T Cell Subsets

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For flow cytometric analysis, the following
antibodies were purchased from BioLegend: CD2-BV510 (clone RPA-2.10),
CD3-FITC (clone HIT3a), CD4-APC/Cy7 (clone RPA-T4), CD8-PE/Cy7 (clone
SK1), CD25-PE (clone M-A251), CD69-PE/Cy5 (clone FN50), PD-1-PerCP/Cy5.5
(clone EH12.2H7). Dead cells were stained using the Zombie Violet
Fixable Viability Kit (BioLegend). Staining was performed in PBS or
PBS supplemented with 1% BSA and T cells were fixed in FluoroFix Buffer
(BioLegend) before flow cytometric acquisition. Fluorescence was measured
using a CyAn ADP Analyzer instrument (Beckman Coulter, The Netherlands)
and data was analyzed using FlowJo (version 10.7.1, Tree Star, Inc.,
USA) to obtain mean fluorescence intensity (MFI; geometric mean),
population frequencies, and proliferation data (division index, average
number of cell divisions). Normalized MFI (NMFI) was calculated by
multiplying the frequency of positive cells by the MFI within the
positive population.

Wauters A.C., Scheerstra J.F., Vermeijlen I.G., Hammink R., Schluck M., Woythe L., Wu H., Albertazzi L., Figdor C.G., Tel J., Abdelmohsen L.K, & van Hest J.C. (2022). Artificial Antigen-Presenting Cell Topology Dictates T Cell Activation. ACS Nano, 16(9), 15072-15085.

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Multicolor Flow Cytometry Analysis of Tumor-Infiltrating Lymphocytes

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Tumors were incubated with collagenase/DNase for 15 min, homogenized and first incubated with Fc Block™ (BD-Biosciences) for 10 min. Then, tumor samples were stained with the following antibodies to simultaneously quantify lymphocyte subsets, OVA(257-264) specific CD8 T-cells and expression of immune checkpoint molecules: CD45-APC-Cy7, K^b/OVA(257-264)-APC, CD4-Alexa 700, CD8-PE, CD3-BUV496, PD1-PerCP/Cy5.5, LAG3-BV650 and TIM3-BV785 (all from Biolegend, San Diego, CA, USA, except CD3-BUV496 which is from BD-Biosciences).
To analyze effector functions cells were stimulated for 4 h with PMA (100 ng/mL)/ionomycin (1 μg/mL) and then stained with antibodies against CD45-APC-Cy7, CD4-Alexa 700, CD8-BV421 CD107a-FITC (all from Biolegend), CD3-BUV496 (from BD-Biosciences). Then cells were fixed and permeabilized and finally stained intracellularly with anti-IFN-γ-PE and TNF-α-BV510.
In all cases the promofluor 840 (maleimide, Promokine, Burlingame, CA, USA) was added to stain dead cells. Samples were acquired with FACSCantoII (Becton Dickinson, Franklin Lakes, NJ, USA) or with Cytoflex (Beckman Coulter, Irving, TX, USA) flow cytometers and analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).

Silva L., Egea J., Villanueva L., Ruiz M., Llopiz D., Repáraz D., Aparicio B., Lasarte-Cia A., Lasarte J.J., Ruiz de Galarreta M., Lujambio A., Sangro B, & Sarobe P. (2020). Cold-Inducible RNA Binding Protein as a Vaccination Platform to Enhance Immunotherapeutic Responses against Hepatocellular Carcinoma. Cancers, 12(11), 3397.

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Multiparametric Flow Cytometry for Expanded TILs

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Expanded TIL were first washed in FACS Wash Buffer (Dulbecco’s phosphate-buffered saline 1X (PBS, Thermo Fisher Scientific)) with 1% bovine serum albumin (Millipore Sigma). Surface Fc receptors were blocked for 10 min at room temperature using goat serum (Sigma) diluted in FACS Wash Buffer (5%) before proceeding with surface staining on ice (100 µL per reaction) for 30 min. Cell surface expression assessment for this study was done using fluorochrome-conjugated antibodies against CD3 FITC (SK7), CD4 PerCP-Cy5.5 (RPA-T4), CD4 BUV496 (SK3), CD28 PE-Cy7 (CD28.2), CD8 PB (RPA-T8) (all BD Bioscience, San Jose, California, USA), LAG3 PE (3DS223H) (Life Technologies, Carlsbad, California, USA), PD-1 PerCP-Cy5.5 (EH12.2H7), CD27 APC (M-T271), CD8 APC-Cy7 (SK1) (Biolegend, San Diego, California, USA). Aqua or Yellow Live/Dead viability stain (Thermo Fisher Scientific) was used to exclude dead cells from analysis. Stained cells were fixed with 1% paraformaldehyde (Electron Microscope Sciences, Hatfield, Pennsylvania, USA) solution for 20 min at room temperature.

Shah P., Forget M.A., Frank M.L., Jiang P., Sakellariou-Thompson D., Federico L., Khairullah R., Neutzler C.A., Wistuba I., Chow C.W., Long Y., Fujimoto J., Lin S.Y., Maitra A., Negrao M.V., Mitchell K.G., Weissferdt A., Vaporciyan A.A., Cascone T., Roth J.A., Zhang J., Sepesi B., Gibbons D.L., Heymach J.V., Haymaker C.L., McGrail D.J., Reuben A, & Bernatchez C. (2022). Combined IL-2, agonistic CD3 and 4-1BB stimulation preserve clonotype hierarchy in propagated non-small cell lung cancer tumor-infiltrating lymphocytes. Journal for Immunotherapy of Cancer, 10(2), e003082.

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