Pe cy7 conjugated anti cd3

Manufactured by BioLegend

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PE-Cy7-conjugated anti-CD3 is a fluorescently-labeled monoclonal antibody that binds to the CD3 protein, which is a component of the T cell receptor complex. The PE-Cy7 fluorophore is attached to the antibody, allowing for detection and analysis of CD3-expressing cells using flow cytometry.

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Anti-CD3 (468 citations) CD3-PE-Cy7 (67 citations) CD3-PE (53 citations) Anti-CD3-PE-Cy7 (52 citations) Anti-CD3-PE (39 citations) PE-conjugated anti-CD3 (9 citations) PE/Cy7-conjugated anti-human CD3 (4 citations) PE/Cy7)-conjugated anti-mouse CD3 (3 citations) PE/Cy7‐conjugated CD3 (2 citations) PE-Cy7-conjugated anti-CD3 (145-2C11) (2 citations)

6 protocols using pe cy7 conjugated anti cd3

Mouse Kidney Cell Isolation and Immunophenotyping

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Mouse kidney tissue was diced and digested with Collagenase-I at 37°C for 1 hour. The suspension was filtered through a 40 um strainer to remove cell pellets and washed in PBS to generate a single cell suspension after lysis of red blood cells (BD, USA). The cells were washed twice with PBS. The third generation of hAD-MSCs were trypsinized and washed twice with PBS. The cells were then incubated with the following fluorescent antibodies and corresponding isotype controls (Cat.Number: 400605, 400611 and 400507, Biolegend, USA) for 30 minutes shielded from light at room temperature: FITC-conjugated anti-CD45, APC-conjugated anti-CD11b, PE-Cy7-conjugated anti-CD3, PE-conjugated anti-F4/80, APC-conjugated anti-CD34, PE-conjugated anti-CD31, FITC-conjugated anti-CD90, APC-conjugated anti-CD44 and PE-conjugated anti-105 (Biolegend, USA). The positive cells were sorted using a BD FACS and the data were analysed using the FlowJo v7.6.3 software.

Zhu F., Chong Lee Shin O.L., Pei G., Hu Z., Yang J., Zhu H., Wang M., Mou J., Sun J., Wang Y., Yang Q., Zhao Z., Xu H., Gao H., Yao W., Luo X., Liao W., Xu G., Zeng R, & Yao Y. (2017). Adipose-derived mesenchymal stem cells employed exosomes to attenuate AKI-CKD transition through tubular epithelial cell dependent Sox9 activation. Oncotarget, 8(41), 70707-70726.

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Assaying CD8+ T Cell Activation

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After transfection (24 h), primary CD8⁺T cells were stimulated using anti-CD3/CD28 (3 μg/ml) for 24 h. The protein transport inhibitor (GolgiStop; 1 μl/ml, BD Biosciences) was added to the culture for the last 6 h. The cells were stained with PE-cy7-conjugated anti-CD3 and APC-cy7-conjugated anti-CD8 (Biolegend). Subsequently, intracellular staining was performed by incubating the cells in 1X Perm/Wash Buffer for 15 min in the dark, followed by incubation with APC-conjugated anti-IFN-γ and FITC-conjugated anti-granzyme B for 30 min at 4°C. After staining, the cells were fixed in 1% formaldehyde. The intracellular expression of IFN-γ and granzyme B was determined using a BD LSR II flow cytometer and data were analyzed using the FlowJo software.

Yin L.B., Song C.B., Zheng J.F., Fu Y.J., Qian S., Jiang Y.J., Xu J.J., Ding H.B., Shang H, & Zhang Z.N. (2019). Elevated Expression of miR-19b Enhances CD8+ T Cell Function by Targeting PTEN in HIV Infected Long Term Non-progressors With Sustained Viral Suppression. Frontiers in Immunology, 9, 3140.

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Profiling Lymphocyte Activation and Th17 Cells

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Antibodies used for flow cytometric analysis (BioLegend) included Fc block (93), PE Cy7-conjugated anti-CD3 (145-2C11), PE-conjugated anti-CD19 (6 days), FITC-conjugated anti-CD69 (H1.2F3), FITC-conjugated anti-IL17a (TC11-18H10.1), and PE-conjugated anti-CD4 (GK1.5). In short, popliteal lymph node cells (PopLN—10⁶ cells in 25 µL) from rechallenged BL6 mice were incubated with FC receptor antibodies (5–10 min) and incubated with conjugated antibodies (20–30 min at 4 °C). After incubation with conjugated antibodies, cells were washed twice with 1× PBS/2% fetal bovine serum buffer. The stained cells were then assessed by flow cytometer (FC500; Beckman Coulter) and the resulting data analyzed by Cytomics CXP software (Beckman Coulter). Intracellular Cytokine Staining Kit (BD biosciences) was used for IL-17a staining per manufacturer’s specifications. Briefly, cells were first stained for cell surface CD4 receptor and then fixed/permeablized. Fixed/permeabolized cells were then stained for IL-17a. Three-color flow cytometric analysis was used to profile the lymphocyte activation. Two-color flow cytometric analysis was used to look for Th17 cell induction.

Sido J.M., Jackson A.R., Nagarkatti P.S, & Nagarkatti M. (2016). Marijuana-derived Δ-9-tetrahydrocannabinol suppresses Th1/Th17 cell-mediated delayed-type hypersensitivity through microRNA regulation. Journal of molecular medicine (Berlin, Germany), 94(9), 1039-1051.

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Apoptosis and activation of CD8+ T cells

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After transfection (72 h), Jurkat cells were stained with PE-conjugated anti-Annexin V and 7-AAD (Biolegend). After transfection (24 h), primary CD8⁺T cells were stimulated using anti-CD3/CD28 (3 μg/ml). After stimulation (48 h), CD8⁺T cells were stained with PE-cy7-conjugated anti-CD3, APC-cy7-conjugated anti-CD8, PE-conjugated anti-Annexin V, and 7-AAD (Biolegend). The cells were analyzed using a BD LSR II flow cytometer and the FlowJo software.

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Comprehensive FACS Staining Protocol

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The following antibodies were used for FACS staining: PE/Cy7 conjugated anti-CD3 (clone UCHT1), PerCP conjugated anti-CD8 (clone RPA-T8), APC conjugated anti-CD45RA (clone HI100), Alexa Fluor 488 conjugated anti-CCR7 (clone G043H7), PE/Dazzle 594 conjugated anti-CD19 (clone HIB19), Alexa Fluor 700 conjugated anti-CD14 (clone HCD14), PerCP conjugated anti-CD19 (clone HIB19), Alexa Fluor 488 conjugated anti-CD20 (clone 2H7), Brilliant Violet 510 conjugated anti-CD38 (clone HB-7), PE/Cy7 conjugated anti-CD24 (clone ML5), Brilliant Violet 421 conjugated CD27 (clone M-T271), Alexa Fluor 647 conjugated anti-IgD (clone IA6-2), PE conjugated anti-SLAMF1/CD150 (clone A12), PE conjugated anti-SLAMF2/CD48 (clone BJ40), PE conjugated anti-SLAMF3/CD229 (clone HLy-9.1.25), PE conjugated anti-SLAMF4/CD244 (clone C1.7), PE conjugated anti-SLAMF5/CD84 (clone CD84.1.21), PE conjugated anti-SLAMF6/CD352 (clone NT-7), PE conjugated anti-SLAMF7/CD319 (clone 162), APC/Brilliant Violet 421/PE conjugated Mouse IgG1 Isotype Control (clone MOPC-21) were purchased from Biolegend. PerCP eFLuor 710 conjugated anti-CD4 (clone SK3) was purchased from eBioscience.

Karampetsou M.P., Comte D., Kis-Toth K., Kyttaris V.C, & Tsokos G.C. (2017). Expression patterns of signaling lymphocytic activation molecule family members in peripheral blood mononuclear cell subsets in patients with systemic lupus erythematosus. PLoS ONE, 12(10), e0186073.

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Intracellular IFN-γ Expression in γδT Cells

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After 12–15 days’ culture, the intracellular IFN-γ expression assay was performed using flow cytometry. γδT cells were incubated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA), 250 ng/mL ionomycin, and monensin (6 μg/mL) for 4 h at 37°C in 5% CO₂. Then, γδT cells were collected, and the following antibodies were used for intracellular staining: PE-Cy7-conjugated anti-CD3 (BioLegend), PE-conjugated anti-TCRγδ (BD Pharmingen), and APC-conjugated anti-IFN-γ (BioLegend). We used a Cytofix/Cytoperm Plus Kit (BD Pharmingen) for intracellular staining, according to the manufacturer’s protocols.

He Y., Xu L., Feng J., Wu K., Zhao Y, & Huang H. (2020). HDAC Inhibitor LBH589 Suppresses the Proliferation but Enhances the Antileukemic Effect of Human γδT Cells. Molecular Therapy Oncolytics, 18, 623-630.

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