Anti cd25 clone pc 61

Manufactured by BioXCell

Sourced in United States

The Anti-CD25 (clone PC-61.5.3) is an antibody used for flow cytometry and cell sorting applications. It binds to the CD25 antigen, which is expressed on activated T cells, regulatory T cells, and other cell types. The antibody can be used to identify and isolate these CD25-positive cell populations.

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13 protocols using anti cd25 clone pc 61

Engineering Anti-αvβ8 Antibody Reagents

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Anti-CD25 (clone PC-61.5.3) and rat isotype control (HRPN, BP0088) were obtained from Bio X Cell. Anti-β8 (C6D4) is a highly specific engineered recombinant antibody to the specificity determining loop 2 (SDL2) domain of αvβ8 consisting of humanized V genes and CH1 domains, with murine linker and CH2/3 domains in an IgG2a format and is produced in CHO-K1 cells (21 , 29 (link)). The αvβ8 and αvβ3 ecto-domains were expressed and purified as previously described (43 (link), 60 ). For all other reagents, see Supplementary Materials and Methods.

Seed R.I., Kobayashi K., Ito S., Takasaka N., Cormier A., Jespersen J.M., Publicover J., Trilok S., Combes A.J., Chew N.W., Chapman J., Krummel M.F., Lou J., Marks J., Cheng Y., Baron J.L, & Nishimura S.L. (2021). A tumor-specific mechanism of Treg enrichment mediated by the integrin αvβ8. Science immunology, 6(57), eabf0558.

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Evaluating PD-1 Blockade and CD25+ Depletion

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For PD-1 blockade experiments, 10-week-old single TCR T cell and WT NOD female mice were treated intraperitoneally (IP) with 250 μg anti–PD-1 (clone J43, Bio X Cell, West Lebanon, NH) on day 0 and with 200 μg on days 2, 4, 6, 8, and 10 (22 (link)). For transient depletion of CD25 positive cells, a single dose of 500 μg anti-CD25 (clone PC-61.5.3, Bio X Cell) was administered intraperitoneally at between 3.5–4 weeks of age (23 (link)).

Schuldt N.J., Auger J.L., Spanier J.A., Martinov T., Breed E.R., Fife B.T., Hogquist K.A, & Binstadt B.A. (2017). Dual TCRα expression poses an autoimmune hazard by limiting Treg cell generation. Journal of immunology (Baltimore, Md. : 1950), 199(1), 33-38.

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CD4+ and CD8+ T-cell Depletion for Pancreatic Tumor Models

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For CD4⁺ and CD8⁺ T-cell depletion studies, 200 μg of anti-CD4 (Bio X cell, BP0003–1, clone GK1.4) and 200 μg of anti-CD8 (Bio X Cell, BP0004–1, clone 53–6.7) or an IgG isotype control rat IgG2b, κ and rat IgG2a, κ (Bio X Cell), respectively, were administered intraperitoneally daily starting 3 days prior to tumor cell injection and twice a week after tumor cell injection. Tregs were depleted using 500 μg of anti-CD25 clone PC 61.5.3 (BioXcell, BE0012), injected i.p. on day 3 and day 1 before pancreatic tumor implantation and repeated every 5 days for the duration of the experiment. Control mice received rat IgG1λ (BioXcell) isotype control. Mice were sacrificed 21 days after tumor implantation, tumor size and weight were measured, and spleen and tumor samples were collected for further processing. Depletion of cells was confirmed by flow cytometry at the end of the experiment.

Mirlekar B., Michaud D., Lee S.J., Kren N.P., Harris C., Greene K., Goldman E.C., Gupta G.P., Fields R.C., Hawkins W.G., DeNardo D.G., Rashid N.U., Yeh J.J., McRee A.J., Vincent B.G., Vignali D.A, & Pylayeva-Gupta Y. (2020). B cell–derived IL35 drives STAT3-dependent CD8+ T-cell exclusion in pancreatic cancer. Cancer immunology research, 8(3), 292-308.

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Immune Cell Depletion Effects on Anti-Tumor Therapy

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The tumor model and treatment regimen were as in the antitumor activity assay but mice were depleted of CD4⁺ T cells using anti-mouse CD4 monoclonal antibody (clone GK1.5, BioXCell, L'Aigle, France), CD8⁺ T cells using anti-CD8β (clone H35-17.2, in house), NK cells using anti-mouse NK1.1 (clone PK136, BioXCell) or conventional CD4⁺CD25⁺Foxp3⁺ T regulatory cells using anti-CD25 (clone PC-61.5.3, BioXCell). InvivoMab rat IgG2b (clone LTF-2, BioXCell) was used as control. A total of 200 µg of each antibody per mouse were administered intraperitoneally 1 day before first therapeutic treatment administration and on days 2, 6, 9, and 13 after it to maintain immune cell depletion during the course of the experiment. B cells were depleted using 500 µg per mouse of InvivoMab anti-CD19 (clone BE0150, BioXCell) 1 day before first therapeutic treatment administration and on days +2, +7, +12, +17, +22 and +27. The effect of IL10 was neutralized using 250 µg/mice of InvivoMab anti-IL10R (CD210) (clone 1B1.3A, BioXCell) administered after the first therapeutic treatment administration and on days +7, and +14.

Tenesaca S., Vasquez M., Alvarez M., Otano I., Fernandez-Sendin M., Di Trani C.A., Ardaiz N., Gomar C., Bella A., Aranda F., Medina-Echeverz J., Melero I, & Berraondo P. (2021). Statins act as transient type I interferon inhibitors to enable the antitumor activity of modified vaccinia Ankara viral vectors. Journal for Immunotherapy of Cancer, 9(7), e001587.

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Multicolor Flow Cytometry of Immune Checkpoint Targets

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Fluorescently-conjugated antibodies CD3-e450, CD8-PerCP, CD4-FITC, CD4-e450, CD4-PerCP, CD25-APC, were purchased from Ebioscience (San Diego, CA). CD8-PE-TxRD was purchased from Invitrogen (Carlsbad, CA). Therapeutic anti-CTLA4 (clone 9D9 or UC10), anti-OX40 (clone OX86), anti-CD40 (clone FGK4.5), anti-CD4 (clone GK1.5), and anti-CD25 (clone PC.61.5.3) antibodies were obtained from BioXcell (Branford, CT) and resuspended in sterile PBS to a concentration of 1mg/mL. Antibodies were administered as 250μg (anti-OX40 and anti-CTLA4) or 100μg (anti-CD4 and anti-CD25) intraperitoneally. DEC205ova was kindly provided by CellDex Therapeutics (Hampton, NJ). SIINFEKL-Kb tetramers were obtained from the NIH Tetramer Core Facility at Emory University (Atlanta, GA).

Young K.H., Baird J.R., Savage T., Cottam B., Friedman D., Bambina S., Messenheimer D.J., Fox B., Newell P., Bahjat K.S., Gough M.J, & Crittenden M.R. (2016). Optimizing Timing of Immunotherapy Improves Control of Tumors by Hypofractionated Radiation Therapy. PLoS ONE, 11(6), e0157164.

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Depleting CD4+, CD8+, and CD25+ Cells in Mice

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Mice were intraperitoneally injected total three times with 0.25 mg/mouse of anti-CD4 (clone GK1.5), anti-CD8 (clone 53.6.72) or anti-CD25 (clone PC-61.5.3) monoclonal antibodies (mAbs), all from Bio X Cell (Lebanon, NH), on the day of tumor inoculation (day 0) and days 4 and 8. Depletions of the corresponding cell populations using this protocol were confirmed by a flowcytometric analysis of spleen cells obtained one day after the mAb inoculation (data not shown). Two independent experiments with 5 mice per group were performed.

Yokomizo K., Waki K., Ozawa M., Yamamoto K., Ogasawara S., Yano H, & Yamada A. (2022). Knockout of high-mobility group box 1 in B16F10 melanoma cells induced host immunity-mediated suppression of in vivo tumor growth. Medical Oncology (Northwood, London, England), 39(5), 58.

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Tumor Growth and Depletion Assays

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To generate subcutaneous tumors, 0.5 × 10⁶ EL4, B16F0, or B16F10 cells were injected subcutaneously (s.c.) into each mouse. Tumor size was measured every other day with a digital caliper. Tumor volume was calculated using the formula [(small diameter)² × (large diameter) × 0.5]. For cell depletion experiments, 200 μg anti-Gr-1 (clone RB6–8C5, BioXcell), anti-CD25 (clone PC-61.5.3, BioXcell), or rat IgG2b isotype control was administered intraperitoneally to each mouse, once every three days, from Day 0 to the last day of the experiment. For c-Rel inhibitor experiments, mice received daily intraperitoneal injections of 10 mg/kg of c-Rel inhibitor R96A³⁹ (in 50 ul DMSO) or DMSO vehicle control. For PD-1 antibody experiments, each mouse received intraperitoneal injections of 100 μg anti-mouse PD-1 (clone J43, BioXcell) or Armenian hamster IgG control (clone N/A, BioXcell), once every three days.

Li T., Li X., Zamani A., Wang W., Lee C.N., Li M., Luo G., Eiler E., Sun H., Ghosh S., Jin J., Murali R., Ruan Q., Shi W, & Chen Y.H. (2020). c-Rel Is a Myeloid Checkpoint for Cancer Immunotherapy. Nature cancer, 1(5), 507-517.

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Immunomodulation in Transplantation

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In the indicated experiments, mice received 500 μg CTLA-4Ig (abatacept, BMS) in 100μL PBS i.p. on the day of transplantation (d0) and days 2, 4, 6 post-transplantation. CD25⁺ T cells were depleted with anti-CD25 (clone PC.61, BioXcell) at a dose of 0.4mg/200μL PBS i.v. on the day of transplantation. Depletion was confirmed by the loss of FoxP3⁺ CD4⁺ T cells in peripheral blood mononuclear cells (PBMCs) 7 days later. anti-PD-L1 blocking antibody (clone B7-H1, BioXcell) was administered on the day of transplantation (d0) at a dose of 0.5mg/200μL PBS and 0.25mg/100μL PBS i.p. days 2, 4 and 6 post-transplantation.

Wang P., Chen L., McIntosh C.M., Lane J.I., Li R., Xie S.Z., Sattar H., Esterhazy D., Chong A.S, & Alegre M. (2022). Oral alloantigen exposure promotes donor‐specific tolerance in a mouse model of minor‐mismatched skin transplantation. American Journal of Transplantation, 22(10), 2348-2359.

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Depletion of regulatory T cells for bacterial infection

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After receiving MSCs intravenously, mice were immediately given i.p. injections of 0.3 mg of anti‐CD25 (clone PC61) or control rat IgG antibodies purchased from Bio X Cell (New Hampshire, United States) as described everywhere.¹⁷ On day 3 post‐antibody treatment, mice were infected with Hi. Two days after infection, bacteria in the lung homogenates were enumerated. Depletion of CD4⁺CD25⁺ cells was confirmed by flow cytometry (Supplementary figure 5c).

Li W., Chen W., Huang S., Yao G., Tang X, & Sun L. (2020). Mesenchymal stem cells prevent overwhelming inflammation and reduce infection severity via recruiting CXCR3+ regulatory T cells. Clinical & Translational Immunology, 9(10), e1181.

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Lgr5-GFP Mice CD8+ T Cell Transfer

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Lgr5-GFP mice were injected with 500 μg of anti-CD25 (clone PC61) or a rat isotype IgG control both from Bio X Cell by tail vein injection. Five days later, depletion was ensured by flow cytometry analysis of CD4 (GK1.5)-FITC and CD25 (PC61)-PerCP-Cy5.5 in blood obtained by clipping the tail. At day five Jedi CD8+ T cells were transferred.

Agudo J., Park E.S., Rose S.A., Alibo E., Sweeney R., Dhainaut M., Kobayashi K.S., Sachidanandam R., Baccarini A., Merad M, & Brown B.D. (2018). Quiescent tissue stem cells evade immune surveillance. Immunity, 48(2), 271-285.e5.

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