Cd25 pc5

Manufactured by Beckman Coulter

Sourced in United States

The CD25-PC5 is a laboratory equipment product from Beckman Coulter. It is used for the detection and analysis of CD25 antigen expression on cells.

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Anti-CD25-PC5 (2 citations)

6 protocols using cd25 pc5

Profiling Regulatory T Cell Phenotypes

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For Treg and related cytokine analyses, stimulated PBMCs were stained to evaluate the expression of markers with CD4-PE-CY7 (Clone SK3) and CD25-PC5 (Beckman Coulter) antibodies (15 min at 4 °C in the dark). After incubation, the cells were fixed, permeabilized, and stained using the eBioscience™ Human Regulatory T Cell Staining Kit (Invitrogen, USA) according to the manufacturer’s instructions. Finally, the cells were stained with intracellular antibodies including BCL-2-FITC (Clone 124), FoxP3-PE (Clone PCH101), IL-10-APC (Clone JES3-9D7), IL-35-AF700 (R&D Systems), and transforming growth factor (TGF)-β-BV421 (Clone TW7-16B4), in accordance with the previous approach outlined in the FCM section.

Liu L., Cheng X., Yang H., Lian S., Jiang Y., Liang J., Chen X., Mo S., Shi Y., Zhao S., Li J., Jiang R., Yang D.H, & Wu Y. (2022). BCL-2 expression promotes immunosuppression in chronic lymphocytic leukemia by enhancing regulatory T cell differentiation and cytotoxic T cell exhaustion. Molecular Cancer, 21, 59.

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Lymphocyte Subsets Evaluation by Flow Cytometry

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Lymphocyte subsets were evaluated within 3 h of blood collection. For Treg detection as CD3⁺CD4⁺CD25⁺CD127^−/low+ cells and CD4⁺ T cell detection, 50 μL of whole blood was stained with a premixed cocktail of conjugated mAbs (Beckman Coulter) for the following markers, CD3-FITC (clone UCHT1), CD25-PC5 (clone B1.49.9), CD4-PC7 (clone 13B8.2), and CD127-PE (clone R34.34) in concentrations according to manufacturer instructions. The gating strategy for CD3⁺CD4⁺CD25⁺CD127^−/low+ cells including details on gating set-up and the analytical and statistical comparability of CD25⁺CD127^−/low+ and CD25⁺FoxP3⁺ quantification approaches are shown in Additional file 1: Figure S1. CD8⁺ cells were detected using 50 μL of whole blood stained with tetraCHROME CD45-FITC/CD4-PE/CD8-ECD/CD3-PC5 multi-color reagent (Beckman Coulter) in concentrations according to the manufacturer instructions. After a 15 min staining for Tregs or CD8⁺ T-cells in the dark, red blood cells were lysed for 15 min in the dark by adding 600 μL of VersaLyse Lysing Solution (Beckman Coulter, France). Cells were subsequently analyzed using a Cytomics FC 500 flow cytometer, hardware compensation and CXP software (Beckman Coulter, USA).

Bencsikova B., Budinska E., Selingerova I., Pilatova K., Fedorova L., Greplova K., Nenutil R., Valik D., Obermannova R., Sheard M.A, & Zdrazilova-Dubska L. (2019). Circulating T cell subsets are associated with clinical outcome of anti-VEGF-based 1st-line treatment of metastatic colorectal cancer patients: a prospective study with focus on primary tumor sidedness. BMC Cancer, 19, 687.

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Quantification of human regulatory T cells

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The cells were washed twice with PBS and stained for 30 min at room temperature in the dark with fluorescent-labeled anti-human monoclonal antibodies mixture: CD4-fluorescein isothiocyanate (FITC), CD25-PC5, and CD127-PE (Beckman Coulter, Brea, CA, United States). Quantification beads (Beckman Coulter, Brea, CA, United States) were used to assess absolute counts of cell subsets. Background fluorescence and nonspecific staining were assessed using appropriate isotype- and fluorochrome-matched control monoclonal antibodies. The frequencies and absolute counts of CD4⁺CD25^highCD127^-/low Tregs and the percentage of Tregs on gated CD4⁺ lymphocytes were assessed by a Cytomics FC 500 flow cytometer (Beckman Coulter, Brea, CA, United States).

Long D., Yu S., Zhang L., Guo Y., Xu S., Rao Y., Huang Z., Luo Q, & Li J. (2022). Increased sIL-2Rα leads to obstruction of IL-2 biological function and Treg cells differentiation in SLE patients via binding to IL-2. Frontiers in Immunology, 13, 938556.

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Phenotyping of NKT cells and Tregs

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NKT-PBMCs and whole peripheral blood samples from healthy controls and CTD-IP patients were stained with the following antibodies: CD3-FITC, CD56-PE, CD127-PE, CD45-ECD, CD4-FITC, CD25-PC5, CD4-FITC/CD8-PE/CD3-PC5, FOXP3–PE, and appropriate isotype controls (Beckman Coulter, Indianapolis, IN, USA). Staining was performed according to the manufacturer’s instructions.

Liu M., Zeng X., Wang J., Fu Z., Wang J., Liu M., Ren D., Yu B., Zheng L., Hu X., Shi W, & Xu J. (2016). Immunomodulation by mesenchymal stem cells in treating human autoimmune disease-associated lung fibrosis. Stem Cell Research & Therapy, 7, 63.

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Flow Cytometry Analysis of T Cell Subsets

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Flow cytometry: T cell subsets were identified using multicolor flow cytometry with standard techniques on the Navios EX flow cytometer (Beckman Coulter, Sykesville, MD, United States). Whole blood samples were drawn from patients at the scheduled time points (T0, T3, and T6), collected in EDTA tubes, and processed for the evaluation of lymphocyte count and their subpopulations. T lymphocyte subsets determined were CD3+CD4+, CD3+CD8+, CD3-CD16+CD56+, CD3+CD4+CD28-, and CD3+CD8+CD28-, using the following monoclonal antibodies: CD3-FITC (clone: UCHT1; Beckman Coulter), CD16 (clone: 3G8; Beckman Coulter), CD56 clone: N901(NKH-1)-PE; Beckman Coulter), CD4-APC (clone: 13B8.2; Beckman Coulter), CD8 PC5.5 (clone: B9.11l Beckman Coulter), CD28-ECD (clone: CD28.2; Beckman Coulter), and CD45-PC7 (clone: J33; Beckman Coulter). Peripheral blood mononuclear cells were obtained by Ficoll density gradient centrifugation. Immunophenotyping of Tregs was performed with the combination of the following monoclonal antibodies: CD45-PC7 (clone: J33; Beckman Coulter), CD4-FITC (clone: 13B8.2; Beckman Coulter), CD25-PC5 (clone: B1.49.9; Beckman Coulter), and FOXP3-PE (clone: 259D; Beckman Coulter).

Vagiotas L., Stangou M., Kasimatis E., Xochelli A., Myserlis G., Lioulios G., Nikolaidou V., Panteli M., Ouranos K., Antoniadis N., Maria D., Papagianni A., Tsoulfas G, & Fylaktou A. (2022). Effect of panel reactive antibodies on T cell immunity reinstatement following renal transplantation. World Journal of Transplantation, 12(10), 313-324.

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Isolation and Culture of Mesenchymal Stem Cells

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Mesenchymal stem cell culture medium (MSCM) was purchased from Sciencell, USA. Lymphocyte separation medium (Ficoll, 1.077 g/mL) was purchased from GE Healthcare Life Sciences, USA. Phytohemagglutinin (PHA) was purchased from Sigma, USA. Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) fluorescent dye was purchased from the VICMED Company, Xuzhou, Jiangsu, China. Mouse anti-human CD34-PE, CD29-PE, CD44-FITC, CD45-FITC, CD127-PE, CD4-FITC, CD25-PC5, CD3-FITC, CD8-PC5, and IL-17A-PE monoclonal antibodies, and the flow cytometer were acquired from Beckman-Coulter, USA.

Guo C.H., Han L.X., Wan M.R., Deng G.J, & Gan J.H. (2015). Immunomodulatory effect of bone marrow mesenchymal stem cells on T lymphocytes in patients with decompensated liver cirrhosis. Genetics and molecular research : GMR, 14(2).

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