Anti mouse cd25 pe pc61

Manufactured by Thermo Fisher Scientific

Anti-mouse CD25 PE (PC61.5) is a fluorescently-labeled antibody that binds to the CD25 antigen, also known as the interleukin-2 receptor alpha chain (IL-2Rα), on the surface of mouse cells.

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Anti-mouse CD25 (PC61.5, (3 citations) Anti-CD25 (PC61.5, (18 citations) Anti-mouse CD25-PE (16 citations) CD25 (PC61.5, (24 citations) Anti-CD25-PE (39 citations) Anti-mouse CD25 (9 citations) CD25-PE (74 citations)

4 protocols using anti mouse cd25 pe pc61

Tumor-Infiltrating Immune Cell Analysis

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Tumor-bearing mice were sacrificed on the last day of treatment. Tumor tissues were minced and digested in collagenase IV (Invitrogen, Carlsbad, CA, USA) and Dnase I (Sigma–Aldrich) for 30 min at 37 °C, and passed through a 70 μm cell strainer. After centrifuging, part of the cells were collected and stained with surface markers antibodies anti-mouse CD45 FITC (30-F11, eBioscience), anti-mouse CD3 PerCP-eFluor710 (17A2, eBioscience), anti-mouse CD8α PE (53–6.7, eBioscience) for 30 min at 4 °C to detect CD4⁺ T cells and CD8⁺ T cells.
Another part of the cells was stained with surface markers antibodies anti-mouse CD45 FITC (30-F11, eBioscience), anti-mouse CD4 APC (GK1.5, eBioscience) and anti-mouse CD25 PE (PC61.5, eBioscience) prior to fixation and permeabilization. Permeabilized cells were then stained with anti-mouse FOXP3 PE-Cy7 (FJK-16s, eBioscience) for 30 min at 4 °C to detect FOXP3⁺ Tregs. The cells were washed and analyzed by a FACS Calibur flow cytometry.

Zhai W., Zhou X., Wang H., Li W., Chen G., Sui X., Li G., Qi Y, & Gao Y. (2020). A novel cyclic peptide targeting LAG-3 for cancer immunotherapy by activating antigen-specific CD8+ T cell responses. Acta Pharmaceutica Sinica. B, 10(6), 1047-1060.

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Quantification of Tumor-Infiltrating T Cells

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Tumour-bearing mice were sacrificed on the last treatment day. Tumour tissues were minced and digested in collagenase IV (Invitrogen, Carlsbad, CA, USA) and Dnase I (Sigmae-Aldrich) for 30 min at 37 °C, and passed through a 70-mm cell strainer. Part of the cells were collected and stained with surface markers antibodies after centrifuging, anti-mouse CD45 FITC (30-F11, eBioscience), anti-mouse CD3 PerCP-eFluor 710 (17A2, eBioscience), and anti-mouse CD8 APC (53e6.7, eBioscience) for 30 min at 4 °C to detect CD4⁺T cells and CD8⁺ T cells.
Another cell part was stained with surface markers antibodies anti-mouse CD45 FITC (30-F11, eBioscience), anti-mouse CD4 APC (GK1.5, eBioscience), and anti-mouse CD25 PE (PC61.5, eBioscience) prior to fixation and permeabilisation. Permeabilized cells were then stained with anti-mouse FOXP3PE-Cy7 (FJK-16 s, eBioscience) for 30 min at 4 °C to detect FOXP3⁺ Tregs. The cells were washed and analysed by a FACS Calibur flow cytometry.

Zhou Y., Song S., Yuan B., Wu Y., Gao Y., Wan G, & Li G. (2022). A Novel CTLA-4 affinity peptide for cancer immunotherapy by increasing the integrin αvβ3 targeting. Discover. Oncology, 13, 99.

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Flow Cytometric Analysis of LTα1β2 in T Cells

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For detection of LTα1β2, T cells were incubated with MOPC21(BioExCel, West Lebanon, NH) or mouse LTβRIg (recognizing both human and mouse LTα1β2, gift from Biogen Idec, Cambridge, MA) at 2 μg /mL for 60 minutes at 37 °C in HBSS with 2% FBS. Cells were washed, and then stained for 45 minutes at 4 °C with anti-CD16/32 (clone 93, eBioscience), followed with BV421-rat anti-mouse IgG1 (Biolegend) APC-eflour780-anti-mouse CD4 (GK1.5, eBioscience), and PE-anti-mouse CD25 (PC61.5, eBioscience); cells were then washed and fixed with 4% paraformaldehyde and run on an LSR Fortessa flow cytometer (BD Biosciences). Surface TLR2 staining used anti-mouse or human TLR2–APC (clone T2.5, BioLegend). Results were analyzed with FlowJo 8.7 (Treestar).

Piao W., Xiong Y., Li L., Saxena V., Smith K.D., Hippen K.L., Paluskievicz C., Willsonshirkey M., Blazar B.R., Abdi R, & Bromberg J.S. (2020). Regulatory T Cells Condition Lymphatic Endothelia for Enhanced Transendothelial Migration. Cell reports, 30(4), 1052-1062.e5.

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Characterizing T Cell Surface Markers

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LECs in phosphate-buffered saline (PBS) containing 0.2 mM calcium, 0.1 mM magnesium, and 0.5% w/v bovine serum albumin (BSA) were treated with anti-CD16/32 (clone 93; eBioscience) to block Fc receptors, and then stained with antibodies to cell surface molecules in the same buffer. Flow cytometry antibody used was: APC-eflour780-anti-mouse CD4 (GK1.5; eBioscience); PE-anti-mouse CD25 (PC61.5; eBioscience); PE-anti-mouse CD44 (IM7; eBioscience); APC-anti-mouse VCAM-1 (clone 647; eBioscience). For detection of LTα2β1, T cells were incubated with MOPC21 or LTβRIg at 2 μg /mL for 60 min at 37 °C in HBSS with 2% FBS. Cells were washed, and then stained for 45 min at 4 °C with BV421-rat anti-mouse IgG1 along with antibodies to other cell surface markers. Cells were then washed and fixed with 4% paraformaldehyde and run on an LSR Fortessa flow cytometer (BD Biosciences, San Jose, CA). Results were analyzed with FlowJo 8.7 (Treestar). Flow cytometry gating information is included in Supplementary Fig 8.

Piao W., Xiong Y., Famulski K., Brinkman C.C., Li L., Toney N., Wagner C., Saxena V., Simon T, & Bromberg J.S. (2018). Regulation of T cell afferent lymphatic migration by targeting LTβR-mediated non-classical NFκB signaling. Nature Communications, 9, 3020.

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