Anti pd 1 pe

Manufactured by BioLegend

Sourced in United States

Anti-PD-1-PE is a fluorescently-labeled antibody that binds to the PD-1 (Programmed Cell Death Protein 1) receptor. PD-1 is an important immune checkpoint molecule that regulates T cell activation and function. The PE (Phycoerythrin) fluorescent label allows for the detection and quantification of PD-1-expressing cells by flow cytometry.

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39 protocols using anti pd 1 pe

Immunophenotyping of Mouse and Human Cells

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For mouse cell phenotype analysis, anti CD3-percp/cy5.5, anti CD4-FITC, anti CXCR5-allophycocyanin, anti ICOS-PE, and anti PD-1-PE were purchased from BioLegend (San Diego, CA, USA). Anti CD19-FITC, anti CD138-PE, anti IgD-allophycocyanin, anti CD27-percp/cy5.5, and relevant IgG isotypes were purchased from eBioscience (San Diego, CA, USA).
For human cell analysis, anti CD3-FITC, anti CD4-percp/cy5.5, anti CXCR5-allophycocyanin, anti PD-1-PE, and anti CD19-PE were purchased from BioLegend. Anti-IL-21 neutralizing antibody (eBioscience) and anti-CD40 (BioLegend) were used for functional analysis in vitro.

Guo J., Zhao C., Wu F., Tao L., Zhang C., Zhao D., Yang S., Jiang D., Wang J., Sun Y., Li Z., Li H, & Yang K. (2018). T Follicular Helper-Like Cells Are Involved in the Pathogenesis of Experimental Autoimmune Encephalomyelitis. Frontiers in Immunology, 9, 944.

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Multiparametric Flow Cytometry Panel

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For flow cytometry, we used FITC anti-CD4, APC anti-CD8, FITC anti-IgM, APC anti-CXCR5, PE anti-PD1, FITC anti-CD62L, PE anti-CD25, APC anti-LAG-3, APC anti-TIM-3, PE anti-PD-1 and FITC anti-ICOS, FITC anti-Annexin V and APC anti-FoxP3 antibodies (all from BioLegend).

Tripathi D., Cheekatla S.S., Paidipally P., Radhakrishnan R.K., Welch E., Thandi R.S., Tvinnereim A.R, & Vankayalapati R. (2018). c-Jun N-terminal kinase 1 defective CD4+CD25+FoxP3+ cells prolong islet allograft survival in diabetic mice. Scientific Reports, 8, 3310.

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Multiparametric Flow Cytometry Analysis of Lymphocyte Subsets

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The following antibodies were used for flow cytometric analysis of lymphocyte subsets and intracellular cytokines: Anti-CD3-APC-H7 (BD Biosciences). Anti-CD4-Brilliant Violet 421, Anti-CD8a-Percp-cyanine 5.5, Anti-CXCR3-Percp-cyanine 5.5, Anti-CCR4-PE, Anti-CCR6-APC, Anti-PD-1- PE (Biolegend), Anti-CD45-FITC, anti-IFNγ-eFluor^®450, Anti-IL4-APC, Anti-IL17-PE (eBioscience), FcR Blocking reagent (Miltenyi Biotec MACS). Fixable viability stain 510, Leukocyte Activation cocktail, with BD Golgiplu (BD Biosciences). Red Blood Cell Lysis Buffer (Solarbio Life Science). Human Lymphocyte Separation Medium (TBD Science). Cytometric Bead Array (BD Biosciences). RPMI1640 (Hyclone).

Lin X., Xu A., Zhou L., Zhao N., Zhang X., Xu J., Feng S, & Zheng C. (2021). Imbalance of T Lymphocyte Subsets in Adult Immune Thrombocytopenia. International Journal of General Medicine, 14, 937-947.

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Spleen and Bone Marrow Flow Cytometry

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In brief, spleens were prepared for flow cytometry by generating single cell suspensions. RBCs were lysed by Tris Ammonium Chloride. Single cell suspensions of bone marrow cells were prepared by flushing the marrow followed by RBC lysis. Resulting single cell suspensions of splenocytes or bone marrow cells were Fc blocked with Trustain FcX (anti-mouse CD16/32 clone 93- Biolegend) and stained with combinations of the following antibodies: anti-B220-PacBlue (RA3–6B2), GL7-FITC, anti-CD95-PeCy7 (Jo2), anti-CD4-Alexa Flour 700 (RM4–5), anti-CXCR5-biotin (2G8), anti-PD-1-PE (29F.1A12), anti-CD11c-BV421 (N418), anti-NK1.1-biotin (PK136), anti-Ly6G-BV711 (1A8), CD86-PeCy5 (GL-1), anti-CD44-BV605/APC (IM7), anti-CD62L-PeCy7 (MEL-14), anti-CD80-PE (16–10A1), anti-CD19-BV605 (6D5), anti-CD267-PE (8F10), anti-CD138-PeCy7 (281–2), and SA-PeCy5 from Biolegend; anti-CD43-FITC (S7), anti-Ly6C-FITC (AL-21), anti-CD11b-AF700 (M1/70), anti-CD90.2-biotin (53–2.1), anti-CD19-biotin (1D3), anti-CD40-FITC (HM40–3), anti-IgD-APC (11–26c.28), and SA-V500 from BD Biosciences. Viability staining was performed by incubating samples with Fixable Viability Dye (e780) from eBioscience. Data were acquired on an LSRII flow cytometer (BD Biosciences) and data were analyzed using FlowJo software (Tree Star).

Schell S.L., Bricker K.N., Fike A.J., Chodisetti S.B., Domeier P.P., Choi N.M., Fasnacht M.J., Luckenbill S.A., Ziegler S.F, & Rahman Z.S. (2021). Context-dependent miR-21 regulation of TLR7-mediated autoimmune and foreign antigen driven antibody-forming cell and germinal center responses. Journal of immunology (Baltimore, Md. : 1950), 206(12), 2803-2818.

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Multi-marker Immune Cell Profiling

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Cells were stained with anti-CD4-APC-CY7, anti-CD8-FITC, anti-CXCR5-APC, anti-PD-1-PE, and anti-ICOS-PECF594 (all from Biolegend), after which they were washed and marker expression was detected using flow cytometry (BD LSR2).

Ye L., Li Y., Tang H., Liu W., Chen Y., Dai T., Liang R., Shi M., Yi S., Chen G, & Yang Y. (2019). CD8+CXCR5+T cells infiltrating hepatocellular carcinomas are activated and predictive of a better prognosis. Aging (Albany NY), 11(20), 8879-8891.

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Comprehensive Immune Profiling by Flow Cytometry

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All flow cytometric analyses were performed using the FACSCanto 10c cytometer (BD Biosciences, Heidelberg, Germany) and BD FACSDiva Software version 8.0.1. Cellular immune status (Panel 1), T cell activation and exhaustion (Panel 2) and B cell phenotype (Panel 3) were determined using specific markers for innate leukocytes, T cells and B cells. Briefly, cell frequencies and total cell numbers in whole blood samples were analyzed on a single-cell platform using TruCount™ tubes (BD Biosciences). Cells were stained for 30 min at room temperature using anti-CD45 allophycocyanin-H7 (APC-H7) or AlexaFluor 700 (AF-700), anti-CD3 fluorescein isothiocyanate (FITC), anti-CD8 allophycocyanin (APC), anti-CD4 peridinin chlorophyll protein complex (PerCP), anti-CD19 AF-700 or Brilliant Violet (BV510), anti-CD56 phycoerythrin (PE), anti-CD14 Brilliant Violet (BV510), anti-CD45RA BV605 and anti-CD62L BV421 or BV510, anti-CD20 APC-cyanine 7 (APC-Cy7), anti-CD27 BV421, anti-CD38 APC, anti-CD24 PerCP, anti-IgD PE, anti-IgM FITC and anti-PD-1 PE monoclonal antibodies (BioLegend and BD Biosciences) either before (Panels 1 and 2) or after (Panel 3) lysis of erythrocytes using 1x Lysing Solution according to the manufacturer’s instructions (Panels 1 and 2: BD Biosciences, Panel 3: Beckman Coulter, Brea, CA, USA).

Bonifacius A., Tischer-Zimmermann S., Dragon A.C., Gussarow D., Vogel A., Krettek U., Gödecke N., Yilmaz M., Kraft A.R., Hoeper M.M., Pink I., Schmidt J.J., Li Y., Welte T., Maecker-Kolhoff B., Martens J., Berger M.M., Lobenwein C., Stankov M.V., Cornberg M., David S., Behrens G.M., Witzke O., Blasczyk R, & Eiz-Vesper B. (2021). COVID-19 immune signatures reveal stable antiviral T cell function despite declining humoral responses. Immunity, 54(2), 340-354.e6.

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Evaluating In Vitro Binding Capacity of Immune Checkpoint Proteins

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Stable cell lines were generated to evaluate in vitro binding capacity of anti-PD-1, CD80-Fc, and 4-1BBL-Fc proteins. For this purpose, total RNA was extracted from anti-CD3/CD28 activated human PBMCs, and the first-strand cDNA was synthesized by Thermo cDNA synthesis kit (Thermo Scientific, USA). Human PD-1 (hPD-1), CTLA-4 (hCTLA-4), CD28 (hCD28), and 4-1BB (h4-1BB) coding genes were amplified by PCR using specific primers and separately inserted into pCHO1.0 expression vector. As described earlier, these constructs were transfected to CHO-K1 by electroporation, and then stable cell lines were produced by puromycin/MTX strategy. The expression of these proteins on the surface of transfected CHO-K1 cells was screened by flow cytometry using anti-PD-1-PE (BioLegend™, USA), anti-CD28-PE (BD Pharmingen™, USA), anti-CTLA4-PE (BioLegend), and anti-4-1BB-PE (BioLegend™, USA) antibodies.

Kaviani E., Hosseini A., Mahmoudi Maymand E., Ramzi M., Ghaderi A, & Ramezani A. (2022). Triggering of lymphocytes by CD28, 4-1BB, and PD-1 checkpoints to enhance the immune response capacities. PLOS ONE, 17(12), e0275777.

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Flow Cytometry Analysis of PD-1 Expression

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Flow cytometry was performed using 50 μL of EDTA-treated peripheral blood incubated for 30 min at 4°C with fluorochrome-labeled monoclonal antibodies (mAbs): anti-CD4-FITC (Beckman), anti-CD8-FITC (Beckman), anti-CD56-FITC (Beckman), and anti-PD-1-PE (BioLegend). Erythrocyte lysis and cell fixation were carried out using OptiLyse C Lysing Solution (Beckman). Treated blood samples passed through the Coulter Epics XL Flow cytometer (Beckman), and the relevant data were acquired and accordingly examined. Data analysis was accomplished by FlowJo software (Tree Star, Ashland).

Jiao Q., Liu C., Yang Z., Ding Q., Wang M., Li M., Zhu T., Qian H., Li W., Tu N., Fang F., Ye L., Zhao Z, & Qian Q. (2014). Upregulated PD-1 Expression Is Associated with the Development of Systemic Lupus Erythematosus, but Not the PD-1.1 Allele of the PDCD1 Gene. International Journal of Genomics, 2014, 950903.

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Comprehensive Immune Phenotyping of Tumor Samples

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Tumor tissue was harvested and cut into small fragments followed by digestion with a tumor disassociation kit (Miltenyi Biotec, USA) for 30 min, and then filtered by 70 μm cell strainers. Cells were stimulated with phorbol myristate acetate (PMA)/ionomyocin in the presence of Golgi stop and Monesin (eBioscience) for 4 h. Cells were washed with PBS and then stained with Live/Dead dye and fluorochrome-labeled antibodies specific to cell surface markers [anti-CD45 Alexa 700 (Clone 30-F11, BioLegend), anti-PD-L1 PE-Cy7 (Clone 10F.9G2, BioLegend), anti-CD3 PerCP (Clone 45-2C11, BioLegend), anti-CD8 APC-Cy7 (Clone 53–6.7, BD), anti-CD11c FITC (ab210308, Abcam), anti-PD1 PE (Clone RMP1–30, BioLegend), anti-CD4 PE-CF594 (Clone RM4–5, BD), anti-MHC-I PE (Clone 28–14-8, BioLegend), anti-CD44 APC-Cy7 (Clone IM7, BioLegend), anti-CD62L APC (Clone MEL-14, BioLegend), anti-CD25 PerCP (Clone 3C7, BioLegend), anti-CD127 FITC (Clone A7R34, BioLegend), anti-ICOS PE-Cy7 (Clone 7E.17G9, BioLegend)]. After fixation–permeabilization, cells were stained with fluoro-conjugated antibodies specific to intracellular markers [anti-Ki67 APC (Clone 16A8, BioLegend), anti-IFNγ APC (Clone XMG1.2, BioLegend), anti-granzyme B FITC (Clone GB11, BioLegend)] or with the isotype control. Flow cytometry analysis was performed on BD LSRFortessa and data were analyzed by FlowJo V10.

Hong S., Bi M., Yu H., Yan Z, & Wang H. (2020). Radiation therapy enhanced therapeutic efficacy of anti-PD1 against gastric cancer. Journal of Radiation Research, 61(6), 851-859.

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Analysis of Gut-Homing B Cells and Tfh Cells

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Immune cells isolated from spleen or mLNs were used for determination of gut-homing B cells and follicular helper T cells (Tfh cells). Gut-homing cells were assayed by anti-B220-FITC, anti-α4β7-APC (Thermo fisher) and anti-CCR9-PE (Biolegend). Tfh cells were stained with anti-CD4-FITC (Biolegend), anti-PD-1-PE (Biolegend) and anti-CXCR5-APC (Biolegend). After staining, cells were analyzed with the Accuri C6 Flow Cytometer System (BD Bioscience).

Hwang H.S., Puth S., Tan W., Verma V., Jeong K., Lee S.E, & Rhee J.H. (2018). More robust gut immune responses induced by combining intranasal and sublingual routes for prime-boost immunization. Human Vaccines & Immunotherapeutics, 14(9), 2194-2202.

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