Antibodies used for cell staining were pre-titrated and used at optimal concentrations. A FACS Aria flow cytometer (Becton Dickinson, USA) and FlowJo software was used for analysis. For FACS purification LCs were stained for CD207 (anti-CD207 PeVio700), CD1a (anti-CD1a VioBlue) and HLA-DR (anti-HLA-DR Viogreen, Miltenyi Biotech, UK). For T cell staining, antibodies anti-CD3 PerCP, anti-CD4 Viogreen, anti-CD127 Pe (Miltenyi Biotech, UK) and anti-CD25 PeCy7 (Invitrogen, UK) were used for surface staining. Anti-FOXP3 FITC (eBiosciences, UK), anti-IL-10 PE (Miltenyi, UK) and anti-IDO1 AlexaFluor647 (Biolegend, UK) antibodies were used for intranuclear and intracellular staining. IDO1 intracellular staining of LCs was performed using Intracellular Fixation & Permeabilization Buffer Set (eBioscience, UK), following manufacturer protocol.
Anti cd25 pe cy7
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-CD25-PE-Cy7 is a flow cytometry reagent that binds to the CD25 antigen, which is expressed on the surface of activated T cells and regulatory T cells. It is conjugated with the fluorescent dye PE-Cy7, allowing for the detection and quantification of CD25-positive cells in a sample. This reagent is commonly used in immunology research and clinical applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd4 fitc, by Thermo Fisher Scientific (2 mentions)
Anti foxp3 fitc, by Thermo Fisher Scientific (1 mentions)
Anti cd4 viogreen, by Miltenyi Biotec (1 mentions)
Intracellular fixation permeabilization buffer set, by Thermo Fisher Scientific (1 mentions)
Anti cd3 percp, by Miltenyi Biotec (1 mentions)
Anti cd207 pevio700, by Miltenyi Biotec (1 mentions)
Anti cd1a vioblue, by Miltenyi Biotec (1 mentions)
Anti hla dr viogreen, by Miltenyi Biotec (1 mentions)
Anti il 10 pe, by Miltenyi Biotec (1 mentions)
Anti cd80 fitc, by Thermo Fisher Scientific (1 mentions)
Anti pd l1 pe, by Thermo Fisher Scientific (1 mentions)
Anti cd8 pe cy5, by Thermo Fisher Scientific (1 mentions)
Lsrii cytometer, by BD (1 mentions)
Anti cd3 alexa 700, by BD (1 mentions)
Anti cd20 ecd, by Beckman Coulter (1 mentions)
Anti eomes efluor 660, by Thermo Fisher Scientific (1 mentions)
Anti cd45ra ecd, by Beckman Coulter (1 mentions)
Anti cd27 apc cy7, by BioLegend (1 mentions)
Anti pd 1 fitc, by Thermo Fisher Scientific (1 mentions)
Anti cd86 apc, by Thermo Fisher Scientific (1 mentions)
7 protocols using anti cd25 pe cy7
1
Multiparameter Flow Cytometry Analysis
2
Comprehensive PBMC Immunophenotyping Protocol
3
Phenotyping CD4+ T Cell Subsets
4
Comprehensive Immune Cell Profiling
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Fluorochrome-coupled antibodies specific for human antigens: anti-CD4-FITC, anti-CD127-PE-Cy7, anti-CD62L-PE, anti-CCR4-PE, anti-CCR7-PE, anti-CCR6-PE, anti-CCR10-PE, anti-CXCR3-PE-Cy5, anti-HLA-DR-FITC, anti-CD25-PE-Cy7, anti-CD39-PE, anti-CD44-PE-Cy7, anti-CD45RA-PE-Cy5, anti-GITR-PE, anti-CD45RO-PE, and anti-CD278-PE (all from eBioscience, USA) were used for cell surface staining. Monoclonal antibodies (mAbs)anti-Foxp3-PE, anti-CTLA-4-PE-Cy7, anti-Helios-FITC, and anti-IDO (indolamine-2,3-dioxygenase)-PE-Cy7 (all from BD Pharmingen, USA) were used for intracellular staining. Quantitative analysis was performed using FlowJo software.
5
Immunophenotyping of Lymphocytes in PCOS
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This experiment was performed following previously described procedures29 (link). Single-cell suspensions from the follicular fluid of infertile women with and without PCOS were adjusted to 0.3 × 106/ml, washed twice in phosphate-buffered saline (PBS) (Invitrogen Life Technologies, Grand Island, NY, USA) and blocked in PBS buffer that contained 1% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. The cells were then stained for 30 min at 4 °C in the dark with conjugated antibodies specific for the following cell surface antigens: anti-CD3 PerCP, anti-CD4 FITC, anti-CD8 PE, anti-CD25 PE-CY7, anti-CD69 APC and anti-PD-1 Brilliant Violet 421 (eBioscience, San Diego, CA, USA). The phenotypic characteristics of the antibody-labeled lymphocytes were analyzed using flow cytometry (Beckman Coulter, Fullerton, CA, USA), and the results were analyzed using FlowJo version 6.0 software (TreeStar Inc., Ashland, OR, USA). Isotype-matched controls were included in each staining protocol.
6
Detecting 4-1BB/4-1BBL and T Cell Activation
7
Immune Cell Preparation Protocol
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DC preparation: iDCs were differentiated from monocytes (Biological Specialty Corporation, Colmar, PA) with the addition of a cocktail of GM-CSF (100 ng/mL) and IL-4 (200 ng/mL) for 6 days at 37 °C, 5% CO 2 . Cytokines were replenished on day 3 by adding fresh medium. For maturation, iDCs were exposed to 1 µg/mL of LPS (Invivogen, cat. tlrl-ebLPS) for 1 day. mDCs were stained using anti-CD83 and anti-CD86 to confirm the purity of the preparation.
PHA blast preparation: Peripheral blood mononuclear cells (PBMCs) were isolated from human whole blood. After 3 days of stimulation with 5 µg/mL of PHA (Sigma-Aldrich, cat. L1668), T cells were further expanded using IL-2 (20 ng/mL) for 4 days. Immunofluorescence staining of anti-CD3, CD4, and CD8 antibodies confirmed the mixed population of the T cells in the PHA blast preparation.
Treg preparation: CD25-positive selection was done using fresh human CD4+ T cells (Biological Specialty Corporation). Tregs (CD4+ CD25+) were expanded using the Miltenyl kit (cat. 130-091-301, San Diego, CA) for 10-14 days. Tregs were frozen using 90% FBS and 10% DMSO. The isolated Tregs were stained with the following antibodies: anti-CD4 AF 488, anti-CD25 PE-Cy 7, and anti-FoxP3 APC (eBioscience, San Diego, CA) and analyzed on an Intellicyte iQue Screener Plus flow cytometer (Albuquerque, NM).
PHA blast preparation: Peripheral blood mononuclear cells (PBMCs) were isolated from human whole blood. After 3 days of stimulation with 5 µg/mL of PHA (Sigma-Aldrich, cat. L1668), T cells were further expanded using IL-2 (20 ng/mL) for 4 days. Immunofluorescence staining of anti-CD3, CD4, and CD8 antibodies confirmed the mixed population of the T cells in the PHA blast preparation.
Treg preparation: CD25-positive selection was done using fresh human CD4+ T cells (Biological Specialty Corporation). Tregs (CD4+ CD25+) were expanded using the Miltenyl kit (cat. 130-091-301, San Diego, CA) for 10-14 days. Tregs were frozen using 90% FBS and 10% DMSO. The isolated Tregs were stained with the following antibodies: anti-CD4 AF 488, anti-CD25 PE-Cy 7, and anti-FoxP3 APC (eBioscience, San Diego, CA) and analyzed on an Intellicyte iQue Screener Plus flow cytometer (Albuquerque, NM).
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