Anti cd25 clone 3c7

Manufactured by BioLegend

Anti-CD25 (clone 3C7) is a monoclonal antibody that binds to the CD25 antigen. CD25 is the alpha subunit of the IL-2 receptor, which is expressed on activated T cells, B cells, and natural killer cells.

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CD25 (clone 3C7, (6 citations) Anti-CD25 (3C7, (4 citations) CD25 (3C7; (3 citations)

3 protocols using anti cd25 clone 3c7

Multi-color Flow Cytometry Protocol

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Multi-color flow cytometry was performed using a BD FACS Canto II (BD bioscience, Germany). Single cell suspensions were obtained from spleen, lymph nodes, lung, blood and tumor and were stained with following antibodies: anti-CD3e-APC (clone 145–2C11, Biolegend), anti-CD4-APC/Cy7 (clone GK1.5, Biolegend), anti-CD8-PerCP (clone 53–6.7, Biolegend), anti-CD25 (clone 3C7, Biolegend), anti-IFNγ-FITC (clone XMG1.2, Biolegend), anti-mouse CD45.1 (APC, Clone A20, eBioscience), anti-mouse CD90.1-PeCy7 (clone OX7, Biolegend), anti-FOXP3-Pacific Blue^TM (clone MF-14, Biolegend) and anti-CCR4-PE/Cy7 (clone 2G12, Biolegend). For isolating tumor-infiltrating lymphocytes, tumors were mechanically disrupted, incubated with 1 mg/mL collagenase and 0.05 mg/mL DNAse (both from Sigma Aldrich) and passed through a cell strainer. Single cell suspensions were layered on a gradient of 44% Percoll (Biochrome, Berlin, Germany) and 67% Percoll prior to centrifugation at 800 g for 30 min. Lymphocytes were obtained from the interphase, were washed with PBS and used for flow cytometry analysis.

Rapp M., Grassmann S., Chaloupka M., Layritz P., Kruger S., Ormanns S., Rataj F., Janssen K.P., Endres S., Anz D, & Kobold S. (2015). C-C chemokine receptor type-4 transduction of T cells enhances interaction with dendritic cells, tumor infiltration and therapeutic efficacy of adoptive T cell transfer. Oncoimmunology, 5(3), e1105428.

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Flow Cytometry Immunophenotyping Protocol

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We used the following BioLegend antibodies: anti-CD3 (clones 17A2 and HIT3a) Pacific-blue, FITC, PE, or APC; anti-CD4 (clones GK1.5 and A161A1) FITC, PE, PerCP-Cy5, PE/Cy7, or APC; anti-CD8 (clone 53-6.7) FITC, APC/Cy7, or APC; anti-CD44 (clone IM7) FITC; anti-CD62L (clone MEL-14) APC; anti-CD25 (clone 3C7) APC; anti-CD69 (clones H1.2F3 and FN50) FITC or PE; anti-CD197 or -CCR7 (clone 4B12) PE/Cy7; anti–IFN-γ (clones XMG1.2 and 4S.B3) APC or PE; anti–IL-12 (clone C15.6) PerCP/Cy5.5; anti–IL-17 (clone TC11-18H10.1) PE-Cy-7; anti–IL-2 (clone JES6-5H4) FITC; anti–TNF-α (clone MP6-XT22) PE or PerCP/Cy5.5; and anti–TGF-β (clone TW7-16B4) APC. Brefeldin A Solution (1000×), Monensin Solution (1000×) (catalog 420701), and Intracellular Staining Permeabilization Wash Buffer (10×) were purchased from BioLegend.

Kumar S., Bhaskar A., Patnaik G., Sharma C., Singh D.K., Kaushik S.R., Chaturvedi S., Das G, & Dwivedi V.P. (2021). Intranasal immunization with peptide-based immunogenic complex enhances BCG vaccine efficacy in a murine model of tuberculosis. JCI Insight, 6(4), e145228.

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Multiparametric Flow Cytometry Analysis of Murine Splenocytes

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Single-cell suspensions from spleen of mice were stained with anti-CD4 (clone GK1.5; BioLegend), anti-CD25 (clone 3C7; BioLegend), and anti-FoxP3 (clone MF-14; BioLegend). In the case of intracellular cytokine staining, splenocytes were cultured in RPMI 1640 medium containing 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin in presence of phorbol-12-myristate-13-acetate (PMA) (50 ng/ml; Sigma), ionomycin (1 μg/ml; Sigma), and monensin (BioLegend) for 5 h. Cells were washed, fixed, permeabilized, and stained with anti-IL-17 (clone TC11-18H10.1; BioLegend). Viable single cells were analyzed based on forward and side light scatter properties with a CyFlow Cube 6 flow cytometer (Sysmex) or MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec).

Tukaj S., Mantej J., Sobala M., Potrykus K., Tukaj Z., Zillikens D., Ludwig R.J., Bieber K, & Kasperkiewicz M. (2021). Therapeutic Implications of Targeting Heat Shock Protein 70 by Immunization or Antibodies in Experimental Skin Inflammation. Frontiers in Immunology, 12, 614320.

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