Antibodies for surface markers staining of DCs and T cells were obtained from BD Biosciences, New York, USA (anti-human CD3-PE, CD3-FITC, CD8-PerCP, CD8-APC, CD56-PE, NKG2D-APC, CD4-FITC, CD4-PerCP, CD107a-FITC, CD25-APC, CD45RO-FITC, CD27-PerCPCY5.5, CD57-APC, CCR7-PE, CD14-APC, CD80-PE, CD83-APC, CD86-FITC, HLA-DR-FITC). Antibodies for intracellular proteins staining were also obtained from BD Biosciences (anti-human IFNγ-APC, TNFα-PECY7, granzyme B-FITC, FoxP3-PE). The intracellular staining was performed by fixing and permeabilizing cells with Cytofix/Cytoperm (BD Biosciences). The experiments were performed by using FACS CantoII (BD Biosciences) flow cytometer, and data were analyzed by using the Flowjo software.
Anti human ifnγ apc
Manufactured by BD
Anti-human IFNγ-APC is a fluorochrome-conjugated monoclonal antibody that binds to human interferon gamma (IFNγ). It is used for the detection and quantification of IFNγ-producing cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 percp, by BD (2 mentions)
Golgistop protein transport inhibitor, by BD (2 mentions)
Anti human il 4 pe, by R&D Systems (2 mentions)
Cytofix cytoperm plus fixation permeabilization kit, by BD (2 mentions)
Foxp3 pe, by BD (1 mentions)
Cd80 pe, by BD (1 mentions)
Cd86 fitc, by BD (1 mentions)
Anti human cd3 pe, by BD (1 mentions)
Cd8 percp, by BD (1 mentions)
Cd56 pe, by BD (1 mentions)
Nkg2d apc, by BD (1 mentions)
Cd107a fitc, by BD (1 mentions)
Cd45ro fitc, by BD (1 mentions)
Cd14 apc, by BD (1 mentions)
Cd83 apc, by BD (1 mentions)
Tnf α pe cy7, by BD (1 mentions)
Granzyme b fitc, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Cd3 fitc, by BD (1 mentions)
5 protocols using anti human ifnγ apc
1
Phenotyping of Immune Cells by Flow Cytometry
2
Cytokine Profiling of T-Lymphocyte Polarization
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Monocyte-derived DCs and macrophages were counted, washed, and then co-cultured with allogeneic peripheral blood lymphocytes (PBL) for three, five, or nine days in RPMI-1640 medium (Sigma-Aldrich) at a moDC/macrophage: T-cell ratio of 1: 10 at 37 °C. To determine which T-lymphocyte populations were polarized by the preconditioned DCs and macrophages after three, five or nine days the T cells were stimulated with 1 µg/ml ionomycin and 20 ng/ml phorbol-myristic acetate (PMA) for 4 h, and the vesicular transport was inhibited by BD GolgiStop™ protein transport inhibitor (BD Biosciences) 12 h before the cell staining. The cells were labeled with anti-human CD4-Peridinin Chlorophyll Protein Complex (PerCP), CD8-PE and/or anti-human CD25-PE-conjugated antibodies (BioLegend). Following this, they were fixed and permeabilized by using BD Cytofix/Cytoperm™ Plus Fixation/Permeabilization Kit (BD Biosciences) and labeled with anti-human IFNγ-APC (BD Biosciences), anti-human IL-4-PE (R&D Systems), anti-human IL-10-Alexa Fluor 488, anti-human IL-17-PE (BioLegend), and anti-human FoxP3-APC (R&D System) antibodies. Fluorescence intensities were measured by FACS Calibur cytometer (BD Biosciences) and data were analyzed by the FlowJo v X.0.7 software (Tree Star).
3
Multicolor Flow Cytometry Immunophenotyping
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For surface marker staining, PBMCs were labeled with the following mAbs: anti-human CD14 PE-Cy7, anti-human CD33 PE, anti-human CD11b FITC, anti-human PD-L1 PerCp-eFluor (eBioscience), anti-human HLA-DR APC, anti-human CD8 PE-Cy7, anti-human CD4 PE (BD Biosciences), anti-human CD3 PB, anti-human CD15 BV421 (Biolegend). After incubation for 20 min at RT, the cells were analyzed using flow cytometer. For whole blood staining, 100 μl of fresh whole blood was labeled with above-mentioned antibodies for 20 min at RT, then lysed with red blood cell (RBC) lysis buffer (BD Biosciences), and subjected to flow cytometry.
For intracellular staining, the cells were fixed and permeabilized using Cytofix/Cytoperm Plus kit (BD Biosciences), and stained with the corresponding intracellular Ab, anti-human IFN-γ APC (BD Biosciences), anti-human IDO PerCp-eFluor (eBioscience), anti-human IL-10 BV421 and anti-human Arg1 PE (Biolegend). Data acquisition and analysis were performed by flow cytometer. Controls for each experiment included cells that were single stained for surface markers or intracellular proteins, unstained cells, and isotype-matched antibodies.
For intracellular staining, the cells were fixed and permeabilized using Cytofix/Cytoperm Plus kit (BD Biosciences), and stained with the corresponding intracellular Ab, anti-human IFN-γ APC (BD Biosciences), anti-human IDO PerCp-eFluor (eBioscience), anti-human IL-10 BV421 and anti-human Arg1 PE (Biolegend). Data acquisition and analysis were performed by flow cytometer. Controls for each experiment included cells that were single stained for surface markers or intracellular proteins, unstained cells, and isotype-matched antibodies.
4
Analysis of T Cell Subsets
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After isolation, PBMCs (3 × 105) were cultured in the presence of anti-CD3/CD28 in a 1:1 ratio and treated with TOFA (0.08–2 μM) for 2 days. To investigate the intercellular cytokines in TCs, hemocytes were stimulated by a leukocyte activator, incorporating ionomycin (500 ng/mL; MilliporeSigma) and PMA (50 ng/mL; MilliporeSigma), then inhibited by brefeldin A (1 μg/mL; MilliporeSigma) in a 5% CO2 environment at 37°C for 4 hours. Thereafter, processed cells were stained with anti-human CD3 Brilliant Violet 421 (BioLegend), anti-human CD8 PE-Cy7 (BioLegend), and anti-human IFN-γ APC (BD Biosciences) for Th1 cell analysis; anti-human IL-17A PE (BioLegend) for Th17 cell analysis; and anti-human FOXP3 PE (BioLegend) for Treg analysis. The labeled cells were measured using a BD Biosciences LSR Fortessa flow cytometer. Ultimately, the harvested data were analyzed with FlowJo software (version 10.0; TreeStar).
5
Cytokine and Chemokine Analysis in moDCs
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Supernatants of moDCs and TU-CM-moDCs were harvested four days after monocyte separation or 24 hours after the stimulation of moDCs. The concentration of IL-6, IL-10, TNFα cytokines, and chemokine IL-8 was measured and validated using OptEIA kits (BD Biosciences) following the manufacturer’s instructions.
To determine which T-lymphocyte populations are responsible for the cytokine production, the T cells were stimulated with 1 μg/ml ionomycin and 20 ng/ml phorbol-myristic acetate (PMA) for 4 hours. The vesicular transport was inhibited by BD GolgiStop™ protein transport inhibitor (BD Biosciences) after the activation. The cells were then labeled with anti-human CD3-FITC, anti-human CD8-PE or CD4-PerCP, and anti-human CD25-PE antibodies (all from BioLegend). The samples were then fixed and permeabilized by BD Cytofix/Cytoperm™ Plus Fixation/Permeabilization Kit (BD Biosciences) and labeled again with anti-human CD8-PE or CD4-PerCP as well as with anti-human IFNγ-APC (BD Biosciences), anti-human IL-4-PE (R&D Systems), anti-human IL-10-Alexa Fluor 488, anti-human IL-17-PE (BioLegend). T cell staining panel is shown inS1 Fig . Fluorescence intensities were measured by Novocyte2000R Flow Cytometer (Agilent (Acea) Biosciences Inc., USA), and data were analyzed by the FlowJo v X.0.7 software (Tree Star).
To determine which T-lymphocyte populations are responsible for the cytokine production, the T cells were stimulated with 1 μg/ml ionomycin and 20 ng/ml phorbol-myristic acetate (PMA) for 4 hours. The vesicular transport was inhibited by BD GolgiStop™ protein transport inhibitor (BD Biosciences) after the activation. The cells were then labeled with anti-human CD3-FITC, anti-human CD8-PE or CD4-PerCP, and anti-human CD25-PE antibodies (all from BioLegend). The samples were then fixed and permeabilized by BD Cytofix/Cytoperm™ Plus Fixation/Permeabilization Kit (BD Biosciences) and labeled again with anti-human CD8-PE or CD4-PerCP as well as with anti-human IFNγ-APC (BD Biosciences), anti-human IL-4-PE (R&D Systems), anti-human IL-10-Alexa Fluor 488, anti-human IL-17-PE (BioLegend). T cell staining panel is shown in
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