Anti cd3 pe cy7

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Anti-CD3 PE-Cy7 is a fluorescently-labeled monoclonal antibody that binds to the CD3 receptor on the surface of T cells. It can be used for the detection and enumeration of T cells in flow cytometry applications.

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Anti-CD3 (512 citations) CD3-PE-Cy7 (37 citations) CD3-PE (37 citations) Anti-CD3-PE (18 citations) Anti-CD3-PE-Cy7 (145-2C11), (4 citations) Anti-human CD3 (UCHT1) PE-Cy7 (3 citations) Hamster anti-CD3 PE-Cy7 (2 citations) PE-Cy7-labeled anti-CD3 (2 citations) PE-Cy7-labeled anti-CD3 (145-2C11), (2 citations)

23 protocols using anti cd3 pe cy7

Vitamin D Modulation of PBMC Phenotype

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In 96-well plates (BD, Franklin Lakes, NJ, USA), 100,000 viable PBMCs/well of 6 Colombian HCs were resuspended in 200 µL RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% FBS and were treated with calcidiol at 250 nM and calcitriol (active form of VitD) at 0.5 nM (both within the physiological range) or with 0.01% v/v EtOH as vehicle control.
Cells were harvested 48 h posttreatments in polypropylene tubes, centrifuged at 700 × g for 5 min and washed with PBS. Extracellular staining was done with fluorochrome-labeled antibodies purchased from eBioscience (Santa Clara, CA, USA): fixable viability dye eFluor 506, anti-CD3 PE-Cy7, anti-CD4 PE, anti-CD8 eFluor^® 450, anti-CD56 PE-Cy7, and anti-TIM-3 APC-Cy7. Samples were acquired on a BD-LSRFortessa™ flow cytometer, and data were analyzed in FACSDiva v.8.0.1 software. The gating strategies are shown in Figure S1 in Supplementary Material.

Aguilar-Jimenez W., Saulle I., Trabattoni D., Vichi F., Lo Caputo S., Mazzotta F., Rugeles M.T., Clerici M, & Biasin M. (2017). High Expression of Antiviral and Vitamin D Pathway Genes Are a Natural Characteristic of a Small Cohort of HIV-1-Exposed Seronegative Individuals. Frontiers in Immunology, 8, 136.

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Spleen and MLN Cell Composition Analysis

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Six days after BMT, uninfected and Hpb-infected mice were sacrificed. The spleen and MLN were isolated for the analysis of cell composition. For surface staining, cells were suspended at 2×10⁷ cells/ml in PBS with 2% FCS, and Fc receptors were blocked with a 2.4G2 mAb (Clone: 93, BioLegend). Antibodies for surface staining were: anti-CD3 FITC, anti-CD3 PE-Cy7 (Clone: 145–2C11), anti-CD4 PE-Cy7 (Clone: GK1.5; eBioscience), anti-H2b PE, anti-H2d PE, and anti-H2b APC (Clones: SF1–1.1, SF1–1.1.1, AF6.88.5; BD Biosciences). For intracellular Foxp3 staining, the Foxp3 staining buffer and anti-Foxp3 PE, Foxp3 PE-Cy7 or Foxp3 APC antibodies (Clone: FJK-16S; eBioscience) were used in accordance with the manufacturer’s instructions. For intracellular GATA3 staining, anti-GATA3 PE (Clone: L50–823; BD Biosciences) or isotype control IgG1, kappa was used.

Li Y., Guan X., Liu W., Chen H.L., Truscott J., Beyatli S., Metwali A., Weiner G.J., Zavazava N., Blumberg R.S., Urban JF J.r., Blazar B.R., Elliott D.E, & Ince M.N. (2018). Helminth-Induced Production of TGFβ and Suppression of Graft-Versus-Host Disease Is Dependent on Interleukin-4 Production by Host Cells. Journal of immunology (Baltimore, Md. : 1950), 201(10), 2910-2922.

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Proliferation Assay for CD8+ T Cells

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T cell proliferation was assessed using carboxyfluorescein succinimidyl ester (CFSE) assays as described previously 37, 38. Briefly, PBMCs from study subjects were thawed and dead cells were removed using magnetic beads (see above). The viable cells were washed with PBS and labelled with CFSE (Molecular Probes, Leiden, the Netherlands) at a final concentration of 5 μM for 10 min. at room temperature. After washing and counting, viable cells were seeded in 96‐well round‐bottom plates at a concentration of 2 × 10⁵ cells/well in the presence or absence of each peptide (10 μg/ml) 38 and incubated for 7 days. Phytohemagglutinin (10 μg/ml) was used as positive control to stimulate T cells.
On day 7, cells were stained 20 min. at 4°C for surface markers with anti‐CD8‐phycoerythrin (PE; eBioscience, San Diego, CA, USA) and anti‐CD3‐PE‐Cy 7 (eBioscience) and analysed by flow cytometry. Gated CD8⁺ T cells were examined for CFSE incorporation in proliferated CD8⁺ T cells. The proportion of proliferating CD8⁺ T cells was determined based on the reduction in CFSE fluorescence over time.

Liu S., Su J., Zhang S., Dong H., Wang H., Luo W., Wen Q., He J., Yang X, & Ma L. (2016). Identification of HLA‐A*11:01‐restricted Mycobacterium tuberculosis CD8+ T cell epitopes. Journal of Cellular and Molecular Medicine, 20(9), 1718-1728.

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Multiparameter Flow Cytometric Phenotyping

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D10-19 M1₅₈ cultures were stained with HLA-A*02:01-M1₅₈ tetramer conjugated to PE or APC at a 1:100 dilution in FACS buffer (PBS with 0.1% bovine serum albumin) or PBS. Cells were then washed twice with cold FACS buffer and stained with a cocktail of antibodies including anti-CD3-PB (Biolegend) or anti-CD3-PeCy7 (eBiosciences), anti-CD8-PerCPCy5.5 or anti-CD8-PerCP (both BD Biosciences), CD27-APC or APC-Cy7 (BD Biosciences), CD45RA-FITC (BD Pharmingen, San Diego, CA, USA) and Live/Dear-NIR (Invitrogen) (Indigenous donors only), washed twice with FACS buffer or PBS. Cells were then resuspended in 200 μl of sort buffer and passed though a 40-μm sieve prior to flow cytometric analysis or sorting.

Clemens E.B., Grant E.J., Wang Z., Gras S., Tipping P., Rossjohn J., Miller A., Tong S.Y, & Kedzierska K. (2015). Towards identification of immune and genetic correlates of severe influenza disease in Indigenous Australians. Immunology and Cell Biology, 94(4), 367-377.

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Isolation and Sorting of Immune Cell Subsets

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Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood using Ficoll Paque Plus (GE Healthcare, Boston, MA, USA) gradient cell separation according to manufacturer’s instructions. Red blood cells were lysed using a 1× dilution of BD Pharm lyse (BD Biosciences) in sterile water and incubating the cells for 5–10 min. For total CD4, CD8, and B cell samples, isolated PBMCs were labeled sequentially with anti-human CD4 and CD8 magnetic beads (Miltenyi Biotec, San Diego, CA, USA) and respective fractions were obtained by positive magnetic selection while B cell-enriched fraction was obtained by negative selection of CD4⁺ and CD8⁺ PBMCs. For CD4 and CD8 naïve and memory populations, isolated PBMCs were labeled with mouse anti-human fluorescent antibodies: anti-CD3 PE/Cy7, anti-CD4 PE, anti-CD8 APC, anti-CD45RA FITC, and anti-CCR7 Pacific Blue (eBiosciences, San Diego, CA, USA). Cells were stained for 30 min and then washed and sorted on a BD FACS Aria II cell sorter. Naïve CD4⁺ and CD8⁺ T cells were sorted based on the CD45RA⁺ CCR7⁺ phenotype. With this sorting strategy, the naïve T cell compartment did not include CD45RA⁺ CCR7⁻ cells that correspond to the exhausted effector memory T cell (T_EMRA), which is often expanded in patients with WAS (Table 1). Cell purity was checked after sorting and was consistently >92%.

O’Connell A.E., Volpi S., Dobbs K., Fiorini C., Tsitsikov E., de Boer H., Barlan I.B., Despotovic J.M., Espinosa-Rosales F.J., Hanson I.C., Kanariou M.G., Martínez-Beckerat R., Mayorga-Sirera A., Mejia-Carvajal C., Radwan N., Weiss A.R., Pai S.Y., Lee Y.N, & Notarangelo L.D. (2014). Next Generation Sequencing Reveals Skewing of the T and B Cell Receptor Repertoires in Patients with Wiskott–Aldrich Syndrome. Frontiers in Immunology, 5, 340.

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Multiparametric Flow Cytometry Immunophenotyping

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Cells were mechanically homogenized, followed by staining for surface molecules using LIVE/DEAD fixable Aqua (Invitrogen, 1:1000 dilution) and anti-CD4 PE-Cy5 (RPA-T4, BD, 1:50 dilution), anti-CD3 PE-Cy7 (UCHT1, eBioscience, 1:50 dilution), anti-CD45RA V450 (H100, eBioscience, 1:100 dilution), anti-CXCR5 conjugated with Alexa Fluor 488 (RF8B2, eBioscience, 1:50 dilution), Alexa Fluor 647 (RF8B2, BD, 1:50 dilution), or biotin (RF8B2, BD Biosciencese, 1:50 dilution)/streptavidin APC-Cy7 (1:400 dilution), anti-PSGL-1 PE (KPL-1, BD, 1:50 dilution) or APC (FLEG, eBioscience, 1:50 dilution), anti-ICOS FITC (C398.4A, eBioscience, 1:50 dilution), anti-CCR7 FITC (150503, R&D Systems, 1:50 dilution), anti-CD62L FITC (DREG56, eBioscience, 1:50 dilution), anti-CXCR4 PE-Cy5 (12G5, eBioscience, 1:50 dilution), anti-CD200 APC (OX104, eBioscience, 1:50 dilution), anti-OX40 PE-Cy5 (ACT35, BD, 1:50 dilution), anti-PD-1 PE-Cy7 (EH12.1, BD, 1:50 dilution), anti-CXCR3 BV421(1C6/CXCR3, BD, 1:50), anti-IL-2RA PE (M-A251, BD, 1:25), anti-CD19 APC-Cy7 (SJ25C1, eBioscience, 1:50 dilution), anti-IgD PE-Cy7 (IA6-2, BD, 1:50 dilution), anti-CD38 V450 (HIT2, BD, 1:50 dilution), and anti-IL-10R PE (3F9, Biolegend, 1:50 dilution).

Kim S.T., Choi J.Y., Lainez B., Schulz V.P., Karas D.E., Baum E.D., Setlur J., Gallagher P.G, & Craft J. (2018). Human Extrafollicular CD4+ T Helper Cells Help Memory B Cells Produce Immunoglobulins. Journal of immunology (Baltimore, Md. : 1950), 201(5), 1359-1372.

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IFNγ Production Assay for KIR3DL1+ NK Cells

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IFNγ production by KIR3DL1⁺ NK cells was assessed essentially as previously described (8 ). Briefly, 5×10⁵ PBMC were incubated with 5x10⁵ target cells (221 transfectants) in the presence of 2500 U IL-2 for 14 hours, with GolgiPlug (BD Biosciences) added one hour into the incubation. Cells were then washed and stained with anti-CD56-APC (BD Bioscience), anti-CD3-PECy7 (eBioscience) and anti-NKB1-FITC (anti-KIR3DL1; BD Bioscience) prior to fixation with 2% paraformaldehyde. Following permeablisation with 0.2% saponin, cells were stained with anti-IFNγ-PerCPCy5.5 (eBioscience) and analysed by flow cytometry. Data was analysed with FlowJo software, with the percentage of IFNγ producing KIR3DL1⁺ NK cells (CD56⁺, CD3⁻) normalised to the maximal IFNγ output when incubated with the parental 721.221 cell line (or 221.B0801 in the analysis of HLA-B*08:01 mutants).

Saunders P.M., Vivian J.P., Baschuk N., Beddoe T., Widjaja J., O'Connor G.M., Hitchen C., Pymm P., Andrews D.M., Gras S., McVicar D.W., Rossjohn J, & Brooks A.G. (2014). The interaction of KIR3DL1*001 with HLA class I molecules is dependent upon molecular microarchitecture within the Bw4 epitope. Journal of immunology (Baltimore, Md. : 1950), 194(2), 781-789.

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Multiparametric Flow Cytometry of Placental Immune Cells

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As described previously,25 (link) placental MNCs were incubated with fluorochrome-conjugated monoclonal antibodies for 40 minutes at 4°C. MNCs were pre-incubated with an anti-human BD Fc blocker (BD Pharmingen, San Diego, CA, USA), followed by staining with the live/dead marker anti-FVD-APC-Cy7 (eBioscience, San Diego, CA, USA). The antibodies used in this study were anti-CD3-PerCP-Cy5.5, anti-CD3-PE-Cy7, anti-CD4-AF700, anti-CD8-PE, anti-CD8-APC, anti-CD28-APC, anti-CD45RA-FITC, anti-CD45RO-PE-Cy7, anti-CD57-FITC, and fixable viability dye-APC-Cy7 (all supplied by eBioscience). MNCs were stimulated with phorbol-myristate acetate/ionomycin/brefeldin A/monensin for 5 hours. Cells were fixed and permeabilized using a Fixation/Permeabilization Buffer kit (eBioscience). The permeabilized cells were washed and resuspended in 1% formaldehyde and further stained for intracellular cytokines with anti-interferon gamma (IFN-γ)-PE-Cy7 and anti-IL-17A-APC. Multicolor flow cytometry was performed using a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA), and data were analyzed using FlowJo software (FlowJo, LLC, Ashland, OR, USA).

Kang Y.E., Yi H.S., Yeo M.K., Kim J.T., Park D., Jung Y., Kim O.S., Lee S.E., Kim J.M., Joung K.H., Lee J.H., Ku B.J., Lee M, & Kim H.J. (2022). Increased Pro-Inflammatory T Cells, Senescent T Cells, and Immune-Check Point Molecules in the Placentas of Patients With Gestational Diabetes Mellitus. Journal of Korean Medical Science, 37(48), e338.

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Comprehensive Immune Cell Profiling

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PBMCs were pre-incubated with an anti-mouse CD16/32 Fc blocker (BD Pharmingen, USA), followed by anti-FVD-APC-Cy7 (all supplied by eBioscience, San Diego, CA, USA) to exclude dead cells. After washing with FACS staining buffer, cells were treated with fluorochrome-conjugated monoclonal antibodies for 40 min at 4°C. The monoclonal antibodies used in this study were as follows: anti-CD3-PerCP-Cy5.5, anti-CD3-PE-Cy7, anti-CD4-AF700, anti-CD8-PE, anti-CD8-APC, anti-CD28-APC, anti-CD45RA-FITC, anti-CD45RO-PE-Cy7, anti-CD57-FITC, anti-TCR gamma/delta-FITC, fixable viability dye-APC-Cy7, anti-interferon (IFN)-γ-PE-Cy7, and anti-tumor necrosis factor (TNF)-α-APC (all supplied by eBioscience, San Diego, CA, USA). For intracellular staining, surface-stained cells were stimulated with phorbol-myristate acetate/ionomycin/brefeldin A/monensin for 5 h, and then fixed and permeabilized using a Fixation/Permeabilization Buffer kit (eBioscience, San Diego, CA, USA). The permeabilized cells were washed and resuspended in 1% formaldehyde and stained with anti-IFN-γ-PE-Cy7 and anti-TNF-α-APC. Multicolor flow cytometry was performed using a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA), and the data were analyzed by FlowJo V10 software (FlowJo, LLC, Ashland, OR, USA).

Ju S.H., Lee E.J., Sim B.C., Nga H.T., Lee H.Y., Tian J., Cho K.J., Park H., Choi D.E., Ham Y.R, & Yi H.S. (2023). Leucine-enriched amino acid supplementation and exercise to prevent sarcopenia in patients on hemodialysis: a single-arm pilot study. Frontiers in Nutrition, 10, 1069651.

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Tumor Dissociation and Flow Cytometry Analysis

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Weighed tumors were minced and allowed to digest in a 2 ml mixture of collagenase (400 U type II collagenase, Worthington) and 0.2 mg/ml DNase I in RPMI media at 37°C for 1 hr. The mixture was gently vortexed every 10-20 minutes. The tissue lysate was filtered through a 40 μm mesh prior to immunostaining. The resulting single cell suspension was stained with fixable viability dye eFluor 780, anti-CD45.2 Pacific Blue, anti-CD3 PE-Cy7, anti-CD3 Alexa Fluor 700, anti-Foxp3 Alexa Fluor 700, anti-CD11c eFluor 615, anti-NK1.1 PE (all from eBioscience), anti-Granzyme B APC and anti-CD4 Qdot 605 (from Life Technologies), anti-CD8 Brilliant Violet 650, anti-CD11b Brilliant Violet 570, anti-CD19 Brilliant Violet 650, anti-F4/80 FITC (all from BioLegend), anti-Ly6C APC, anti-Ly6G PE-Cy7, and anti-Ki67 PE (BD Biosciences). The percent positive cells were analyzed by FlowJo and gated on CD45 positivity. To analyze the number of CD133⁺CD44⁺ cells, the single cell suspension was incubated in the dark, on ice with Aqua LIVE/DEAD Fixable Dead Cell Stain (Molecular Probes) 1:1000 for 30 min, followed by staining with anti-CD44 APC (eBioscience, 1: 400) and anti-CD133 PE (eBioscience, 1: 200). Unstained, LIVE/DEAD only, and single stain served as control. Doublets were gated out using forward scatter width/height and sideward scatter width/height event characteristics.

Özdemir B.C., Pentcheva-Hoang T., Carstens J.L., Zheng X., Wu C.C., Simpson T., Laklai H., Sugimoto H., Kahlert C., Novitskiy S.V., Acosta A.D., Sharma P., Heidari P., Mahmood U., Chin L., Moses H., Weaver V., Maitra A., Allison J.P., LeBleu V.S, & Kalluri R. (2014). Depletion of Carcinoma-Associated Fibroblasts and Fibrosis Induces Immunosuppression and Accelerates Pancreas Cancer with Diminished Survival. Cancer cell, 25(6), 719-734.

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