Cd25 pc61

Manufactured by Thermo Fisher Scientific

Sourced in United States

CD25 (PC61.5) is a monoclonal antibody that recognizes the mouse CD25 antigen, also known as the Interleukin-2 Receptor alpha chain (IL-2Rα). It is a useful tool for the identification and isolation of activated T cells and regulatory T cells (Tregs) in mouse samples.

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Anti-CD25-APC (PC61.5, (6 citations) Anti-CD25-PE-Cy7 (clone PC61.5) (3 citations) Anti-mouse CD25 (clone PC61.5, (3 citations) CD25 APC (PC61.5, (3 citations) CD25 (clone PC61.5, (3 citations) Anti-CD25 APC (clone PC61.5) (2 citations) Biotinylated anti-CD25 mAb (clone PC61, (2 citations) Anti-CD25-PE antibodies (PC61.5, (2 citations) CD25 (PC61.5) (2 citations) Anti-CD25-PE-Cy5 (PC61.5, (2 citations)

24 protocols using cd25 pc61

Comprehensive Immune Cell Analysis

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Spleens were collected and single cell suspensions made. For T helper cell analysis, cells were surfaced stained with anti-CD4 (RM 4-5; eBioscience), permiabalized and intracellular stained with IFN-γ (XMG1.2; eBioscience), IL-4 (BVD6-24G2; eBioscience), RORγ (B2D; eBioscience), CD25 (PC61.5; eBioscience) or FoxP3 (FJK-16s; eBioscience). Representative gating in Supplemental Data Figure S1. For CD8⁺ T cells, cell surfaces were stained with anti- CD8a (53-6.7; eBioscience) and anti- CD3e (145-2C11; eBioscience). For B cells, cell surfaces were stained with anti- CD21/CD35 (8D9; eBioscience) and anti- CD19 (1D3; eBioscience). T cell activation markers CD25 (eBio3C7; eBioscience) and CD69 (H1.2F3, Biolegend) were measured using flow cytometry.
For CBC exams ≈ 50 μL of blood was collected into EDTA coated tubes via retro-orbital bleed. VetScan HM5 Analyzer was used.

Chapman L.M., Ture S.K., Field D.J, & Morrell C.N. (2017). miR-451 Limits CD4+ T-Cell Proliferative Responses to Infection in Mice. Immunologic research, 65(4), 828-840.

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In vivo T cell Immunophenotyping

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In vivo labeling of T cells with fluorescent antibodies was performed as previously described (Turner et al., 2014). Briefly, mice were administered, PE- or Alexa 647-conjugated anti-CD90.2 (anti-Thy1.2; clone 53-2.1, BioLegend), or PE-conjugated anti-CD45.2 antibody (clone 104, Biolegend), intravenously, and 7–10 minutes later lungs were perfused, dissected and digested in RPMI1640 medium with collagenase D, DNAse and Trypsin inhibitor for 1 hour at 37°C. Mediastinal lymph nodes and spleen were dissected and manually disrupted to generate single cell suspensions. Cell suspensions were stained with fluorescent-conjugated antibodies for CD4 (clones RM4-5, BD Biosciences, and GK1.5, eBioscience), CD8 (clone 53-6.7, BioLegend), CD11a (clone M17/4, BioLegend),CD11b (M1/70 eBioscience), CD11c (N418, eBioscience), CD25 (PC61.5, eBioscience), CD45 (30-F11, BioLegend), CD69 (clone H1.2F3, eBioscience), CD86 (GL-1, Biolegend), CD103 (clone 2E7, eBioscience) and Anti-Mouse MHC Class II (I-A/I-E) (clone M5/114.15.2, eBioscience) was performed according to manufacturers’ protocol. Stained cells were analyzed using the BD LSRII flow cytometer and flow-jo software (Treestar, Ashland, OR). Absolute cell numbers were determined by flow cytometry using CountBright Absolute Counting Beads (Invitrogen) according to manufacturer’s protocol.

Turner D.L., Goldklang M., Cvetkovski F., Paik D., Trischler J., Barahona J., Cao M., Dave R., Tanna N., D’Armiento J.M, & Farber D.L. (2018). Biased generation and in situ activation of lung tissue-resident memory CD4 T cells in the pathogenesis of allergic asthma. Journal of immunology (Baltimore, Md. : 1950), 200(5), 1561-1569.

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Characterization of T Cell Subsets

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Mouse specific antibodies against CD3ε/γ (17A2; Biolegend), CD4 (RM4-5; eBioscience), CD25 (PC61.5; eBioscience), CD44 (IM7; BD Biosciences), CD62L (MEL-14; Life Technologies), CD126 (D7715A7; eBioscience), CD127 (25-1271-82; eBioscience),βTCR (H57-597), gp130 (125623; R&D Systems), IFNγ (XMG1.2; eBioscience), IL-4 (11B11; eBioscience), IL-17A (TC11-18H10.1; Biolegend), IL-21 (Recombinant mouse IL-21R Fc Chimera protein; R&D and IL-21 receptor antibody; Jackson Immuno Research) and PTPN2 (AF1930; R&D) were used. For detection of human antigens, we used antibodies specific to CD3 (UCHT1; BioLegend), CD4 (RPA-T4; eBioscience), CD45RA (HI100; BioLegend), CD45RO (UCHL1; BioLegend), CD62L (DREG-56; BD Biosciences), CD197 (CCR7; G043H7; BioLegend). Human and mouse cross-specific antibodies to pY STAT1 (pY701; 4a), pY-STAT3 (pY705; 4/P-STAT3), pS-STAT1 (pS727; K51-856) and pS STAT3 (pS727, 49/p-Stat3) were from BD Biosciences.

Twohig J.P., Figueras A.C., Andrews R., Wiede F., Cossins B.C., Soria A.D., Lewis M.J., Townsend M.J., Millrine D., Li J., Hill D.G., Fernandez J.U., Liu X., Szomolay B., Pepper C.J., Taylor P.R., Pitzalis C., Tiganis T., Williams N.M., Jones G.W, & Jones S.A. (2019). Activation of naïve CD4+ T cells re-tunes STAT1 signaling to deliver unique cytokine responses in memory CD4+ T cells. Nature immunology, 20(4), 458-470.

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Multiparametric Flow Cytometry Analysis of Immune Cells

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Human single cell suspensions were surface stained with fluorescence conjugated mAb to CD134 (L106, BD Bioscience), CD137 (4B4-1, BD Bioscience), CD69 (FN50, eBioscience), CD56 (N901, Beckman Coulter), CD3 (SK7, eBioscience) and CD5 (OKT1, in house). Murine cells were first FcγR-blocked (2.4G2, in-house) and then stained with CD49b (DX5, eBioscience), NKp46 (29A1.4, eBioscience), CD3 (17A2, eBioscience), CD4 (RM4–5, eBioscience), CD8 (53–6.7, eBioscience) or CD25 (PC61.5, eBioscience). For intracellular FOXP3 (NRRF-30, eBioscience) staining, cells were fixed and permeabilised as per manufacturer’s protocol (eBioscience). Acquisition was performed using FACSCalibur (BD Biosciences) and analysed using Cytobank (Cytobank).

Turaj A.H., Cox K.L., Penfold C.A., French R.R., Mockridge C.I., Willoughby J.E., Tutt A.L., Griffiths J., Johnson P.W., Glennie M.J., Levy R., Cragg M.S, & Lim S.H. (2018). Augmentation of CD134 (OX40)-dependent NK anti-tumour activity is dependent on antibody cross-linking. Scientific Reports, 8, 2278.

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Immune Cell Characterization in Lymph Nodes

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Single cells suspensions were prepared from mesenteric lymph nodes (MLN) by forcing the organs through an 80µm mesh (Sigma-Aldrich). Cells were washed twice with PBS and then suspended in Flow Cytometry Staining Buffer (eBioscience). To block unspecific binding of antibodies, cell suspensions were incubated with an anti‐CD16/32 mAb (2.4G2, eBioscience) for 15 min on ice and then stained with combinations of the following fluorochrome-labeled antibodies against surface markers for 30 min on ice: CD3 (17A2, eBioscience), CD4 (GK1.5, eBioscience), CD69 (H1.2F3, Abcam), and CD25 (PC61.5, eBioscience). Intracellular staining for FoxP3 (FJK-16s, eBioscience) was performed using the FoxP3/Transcription factor staining buffer set (eBioscience) according to the manufacturer’s instructions. Flow cytometric analysis was performed using a FACSCalibur based on CellQuest software (BD-Becton Dickinson, Franklin Lakes, USA). T cell effectors (Teff) were defined by: CD3, CD4, CD69+. T cell regulators (Treg) were defined by: CD3, CD4, CD25, FoxP3+.

Tolomeo A.M., Castagliuolo I., Piccoli M., Grassi M., Magarotto F., De Lazzari G., Malvicini R., Caicci F., Franzin C., Scarpa M., Macchi V., De Caro R., Angriman I., Viola A., Porzionato A., Pozzobon M, & Muraca M. (2021). Extracellular Vesicles Secreted by Mesenchymal Stromal Cells Exert Opposite Effects to Their Cells of Origin in Murine Sodium Dextran Sulfate-Induced Colitis. Frontiers in Immunology, 12, 627605.

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Multicolor Flow Cytometry Analysis

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Cells were washed twice and treated with CD16/32 (eBiosciences) for 15 min at 4°C before being surface stained for CD4 GK1.5 (PerCP or FITC), CD8 53-6.7 (FITC or APC), and CD25 PC61.5 (APC) (eBiosciences). Cells were prepared for intracellular staining using eBiosciences FoxP3 staining kit. Following fixation and permeabilization, the cells were treated with 2% rat serum to prevent non-specific binding. Cells were stained for FOXP3 FJK-16s (PE), IFN-γ XMG1.2 (PE), and Ki67 SolA15 (PE) (eBiosciences) for 30 min and acquired using an Accuri C6 flow cytometer (BD Biosciences).

Carty F., Corbett J.M., Cunha J.P., Reading J.L., Tree T.I., Ting A.E., Stubblefield S.R, & English K. (2018). Multipotent Adult Progenitor Cells Suppress T Cell Activation in In Vivo Models of Homeostatic Proliferation in a Prostaglandin E2-Dependent Manner. Frontiers in Immunology, 9, 645.

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Phenotypic Profiling of Immune Cells

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Phenotypic analyses of cell surface markers in spleen, lymph node and thymus were performed on unchallenged mice at 6-8 weeks of age. Spleens were processed into cell suspensions through a 70μm strainer by a syringe plunger in RPMI with 10% FBS and then treated with ACK lysis buffer to remove red blood cells. Lymph nodes were processed using glass sides and the thymi were minced. The resulting cell suspensions were centrifuged and stained with antibodies specific for CD4 (RM4-5), CD8α (53-6.7), CD62L (MEL-14), CD44 (IM7), all BD Pharmingen (San Jose, CA), CD25 (PC61.5, ebioscience, San Diego CA), and a viability stain (Life Technologies, Carlsbad, CA). After staining for 25 minutes at 4°C cells were fixed in PBS containing 2% paraformaldehyde. All flow cytometry data was acquired on an LSRII cytometer (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (Treestar, Ashland, Oregon).

Wesley E.M., Xin G., McAllister D., Malarkannan S., Newman D.K., Dwinell M.B., Cui W., Johnson B.D, & Riese M.J. (2018). Diacylglycerol kinase ζ (DGKζ) and Casitas b-lineage proto-oncogene b–deficient mice have similar functional outcomes in T cells but DGKζ-deficient mice have increased T cell activation and tumor clearance. ImmunoHorizons, 2(4), 107-118.

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Endothelial Cell and Endothelial Microparticle Sorting

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Endothelial cells (ECs) were sorted as Ter119⁻CD41⁻CD144⁺. EMP were sorted as Sca1⁻Ter119⁻CD16/32⁺cKit⁺CD41⁺. The following antibodies were used for these studies with various fluorescent color combinations: CD144 (11D4.1, BD Biosciences; 1:200), CD41 (MWReg30, eBioscience; 1:200), CD16/32 (clone 93, eBioscience; 1:200), Ter119 (TER-119 BD Biosciences; 1:200), CD8a (53-6.7, eBioscience; 1:200), CD4 (GK1.5, eBioscience; 1:200), CD45 (30 F-11, BD Biosciences; 1:200), CD25 (PC61.5, eBioscience; 1:200), CD44 (IM7, eBioscience; 1:200), CD117 (2B8, eBioscience; 1:200), Sca1 (D7, eBioscience; 1:200), CD11b (M1/70, eBioscience; 1:200), CD19 (1D3, eBioscience; 1:200), B220 (RA3-6B2, eBioscience; 1:200), CD93 (AA4.1, eBioscience; 1:200). Cells were sorted on FACSAria II (BD Biosciences).

Azevedo Portilho N., Scarfò R., Bertesago E., Ismailoglu I., Kyba M., Kobayashi M., Ditadi A, & Yoshimoto M. (2021). B1 lymphocytes develop independently of Notch signaling during mouse embryonic development. Development (Cambridge, England), 148(15), dev199373.

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Characterization of T Cell Subsets

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Immune Cell Characterization Protocol

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DNFB, brefeldin A, and PF3644022 were from Sigma-Aldrich; SC409, PD98059, SP600125, and rapamycin from EMD Millipore; CFSE from Life Technologies; IFN-γ, IL-2, IL-4, IL-6, and TGF-β from Peprotech. Fluorescent-conjugated antibodies against the following markers were used in flow cytometry: CD3 (145–2C11), CD4 (GK1.5 and RM4–5), CD8 (53–6.7), CD25 (PC61.5), Foxp3 (anti-mouse/rat staining kit), IFN-γ (XMG1.2), IL-4 (BVD6–24G2), IL-17A (eBio17B7; all from eBioscience). Antibodies against the following proteins were used in cell stimulation, Fc receptor blocking, and cytokine neutralization: CD3 (145–2C11), CD28 (37.51), IFN-γ (XMG1.2), IL-12 (C17.8), IL-4 (11B11), CD16/CD32 (2.4G2; all from BD Pharmingen); and hamster IgG (MP Biomedicals). Antibodies against the following proteins were used in immunoblotting: ERK (9102), phosphorylated (p-) p38 (9211), p-JNK (9251), p-ERK (9101), p-S6K1 (9205), p-S6 (4858), p-MK2 (3007), p-TSC2 (3616; all from Cell Signaling Technology); p38α (sc-535; Santa Cruz Biotechnology); p38β (33–8700; Life Technologies); p38γ and p38δ (University of Dundee); JNK (554285, BD Pharmingen); Foxp3 (14–5773, eBioscience); and actin (A4700; Sigma-Aldrich).

Hayakawa M., Hayakawa H., Petrova T., Ritprajak P., Sutavani R.V., Jiménez-Andrade G.Y., Sano Y., Choo M.K., Seavitt J., Venigalla R.K., Otsu K., Georgopoulos K., Arthur J.S, & Park J.M. (2016). Loss of Functionally Redundant p38 Isoforms in T Cells Enhances Regulatory T Cell Induction. The Journal of Biological Chemistry, 292(5), 1762-1772.

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