We assessed the phenotype of Tregs by flow cytometry. CBMCs and PBMCs were cultured with PHA and the indicated concentration of l -arginine at 37°C for 48 h, then washed with fluorescence-activated cell sorting (FACS) buffer prior to evaluation of their Treg subsets. At the end of the stimulation, the cells were stained with anti-CD4-PerCP (BD Biosciences, Franklin Lakes, NJ, USA) and anti-CD25-FITC (Beckman Coulter, Brea, CA, USA) for 30 min, then fixed with paraformaldehyde/phosphate-buffered saline (PBS), and permeabilized using FACS permeabilizing solution (Sigma-Aldrich, St. Louis, MO, USA). For intracellular staining of FoxP3 and IL-10, cells were stained with anti-human FoxP3-PE (eBioscience, San Diego, CA, USA) and IL-10-APC (BD Biosciences). We analyzed the percentages of human CD4+CD25−, CD4−CD25+, and CD4+CD25+ cells, and the intracellular expression of FoxP3 and IL-10 in the subsets, using a FACSCalibur flow cytometer (BD Biosciences).
Anti cd25 fitc
Manufactured by Beckman Coulter
Sourced in United States
Anti-CD25-FITC is a monoclonal antibody conjugated with the fluorescent dye FITC (Fluorescein Isothiocyanate). It is designed to bind to the CD25 antigen, which is expressed on the surface of activated T cells and regulatory T cells. This product can be used for the identification and enumeration of these cell populations in flow cytometry applications.
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Lab products found in correlation
Anti cd3 pc5, by Beckman Coulter (2 mentions)
Anti cd4 pe, by Beckman Coulter (2 mentions)
Anti human foxp3 pe, by Thermo Fisher Scientific (1 mentions)
Anti cd4 percp, by BD (1 mentions)
Il 10 apc, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Anti cd4 apc, by Beckman Coulter (1 mentions)
Anti cd8 apc, by Beckman Coulter (1 mentions)
Anti cd45ra pe, by Beckman Coulter (1 mentions)
Magnetic microbead, by Miltenyi Biotec (1 mentions)
Facsaria 3 cell sorter, by BD (1 mentions)
Anti cd8 fitc, by Beckman Coulter (1 mentions)
Anti cd4 fitc, by Beckman Coulter (1 mentions)
Anti cd56 pe, by Beckman Coulter (1 mentions)
Anti tcr vγ9 fitc, by Beckman Coulter (1 mentions)
Anti cd45 ecd, by Beckman Coulter (1 mentions)
Anti ifn γ fitc, by Beckman Coulter (1 mentions)
Cytomics fc500, by Beckman Coulter (1 mentions)
Anti cd3 fitc, by Beckman Coulter (1 mentions)
Anti cd4 pc5, by Beckman Coulter (1 mentions)
4 protocols using anti cd25 fitc
1
Phenotyping Treg Subsets by Flow Cytometry
2
Isolation and Sorting of Naive CD4+ T Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient centrifugation. CD4+ T cells were enriched with magnetic microbeads (Miltenyi Biotec). Naive CD4+ T cells were sorted as CD4+ CCR7+ CD45RA+ CD25– CD8– on a FACS Aria III cell sorter (BD Biosciences). For cell staining, the following antibodies were used: anti-CD4-APC (allophycocyanin), clone 13B8.2; anti-CD8-APC, clone B9.11; anti-CD8-FITC (fluorescein isothiocyanate), clone B9.11; anti-CD4-FITC, clone 13B8.2; anti-CD45RA-PE (phycoerythrin), clone alb11; anti-CD25-FITC, clone B1.49.9 (all from Beckman Coulter); anti-CCR7-Brilliant Violet 421, clone G043H7 (Biolegend).
3
Immunophenotyping of Peripheral Blood
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The peripheral blood of patients underwent immunophenotyping before the start of therapy and before the fourth therapy. For flow cytometry analysis, the authors used the following monoclonal antibodies: anti-CD3-FITC, anti-CD3-PC5, anti-CD4-PE, anti-CD8-PC5, anti CD25-FITC, anti-CD45-ECD, anti-CD56-PE, anti-TCRpanδγ-FITC, anti-TCRpanαβ-PE, anti-TCRVγ9-FITC, anti-IFNγ-FITC, anti-IL4-PE (Beckman Coulter, Brea, CA, USA) and anti-Foxp3-PE (Becton Dickinson, Franklin Lakes, NJ, USA). Data were analyzed using Cytomics FC 500 (Beckman Coulter).
4
Comprehensive T Cell Subset Analysis
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Flow cytometry was carried out in order to analyse the T lymphocyte subsets (T. Chen et al. Citation2020). CD3 + CD45 + cells indicated the total T cells. Various T lymphocyte subsets, containing T helper cells (CD3 + CD4 + ), T suppressor cells (CD3 + CD8 + ), and regulatory T cells (Tregs; CD4 + CD25 + CD127 Dim ) were individually detected with an FC500MCL flow cytometer (Beckman Coulter, Chaska, MN, USA) using corresponding antibodies anti-CD3-PC5, anti-CD4-PE, anti-CD8-ECD, anti-CD4-PC5, anti-CD25-FITC, and anti-CD127-PE (all procured from Beckman Coulter). Briefly, 2 mL venous peripheral blood samples were collected into EDTA tubes for cytometric analysis, 50 μL whole blood per tube was incubated with monoclonal antibodies for 30 min in conditions void of light, and thereafter red blood cells were lysed using a buffer comprising 0.8% NH 4 Cl and 0.1% KHCO 3 (pH 7.1-7.4). Following a rinse with phosphate-buffered saline (PBS), the cells were suspended with 200 µL PBS for detection of T lymphocyte subsets. The percentages of CD3 + T cells, CD3 + CD4 + T cells, CD3 + CD8 + T cells, and Tregs were calculated using the BD Multitest software (BD Biosciences, San Jose, CA, USA).
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