CD45-RO + frequencies were assessed directly ex vivo by surface staining at 4 °C in PBS with antiCD45RO-FITC, CD3-PE and CD4-APC (all from BD Biosciences). For post-stimulation cultures, dead cells were labelled with near-IR LIVE/DEAD fixable dead cell stain (Molecular Probes, Life Technologies) before fixation. For analysis of regulatory markers: CTLA-4, Foxp3 and CD25, cells were fixed, permeabilised and stained with ebioscience/Thermofisher Foxp3 staining buffers according to the manufacturer's instructions. For analysis of cytokine expression, PMA/ionomycin-restimulated cells were fixed with 3% paraformaldehyde in PBS for 12 min followed by a 5-minute wash with PBS under centrifugation. Fixed cells were permeabilised with 0.1% saponin (Acros Organics) prepared in PBS and stained with IL-17-PE, IFNγ-e450, IL-21-APC, CD3-PERCP, CD4-FITC. For all studies cells were acquired on a Dako Cyan flow cytometer (Dako Cytomation) and data analysed using FlowJo software (Tree Star version 8.8.6). All antibodies were purchased from ebioscience/Thermofisher or BD Biosciences and expression quantified relative to the appropriate isotype control.
Il 17 pe
Manufactured by Thermo Fisher Scientific
Sourced in United States
IL-17-PE is a fluorescently-labeled antibody that binds to the interleukin-17 (IL-17) protein. It is commonly used in flow cytometry applications to identify and quantify IL-17-producing cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 fitc, by Thermo Fisher Scientific (7 mentions)
Foxp3 pe, by Thermo Fisher Scientific (4 mentions)
Cd25 pe, by Thermo Fisher Scientific (4 mentions)
Ifn γ pe, by Thermo Fisher Scientific (3 mentions)
Ifn γ apc, by Thermo Fisher Scientific (3 mentions)
Anti β actin antibody, by Merck Group (3 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Foxp3 pe cy7, by Thermo Fisher Scientific (3 mentions)
Dako cyan flow cytometer, by Agilent Technologies (2 mentions)
Cd4 apc, by BD (2 mentions)
Cd3 pe, by BD (2 mentions)
Saponin, by Thermo Fisher Scientific (2 mentions)
Live dead fixable near ir dead cell stain, by Thermo Fisher Scientific (2 mentions)
Anti cd45ro fitc, by BD (2 mentions)
Cd4 pe, by Thermo Fisher Scientific (2 mentions)
Il 4 pe, by Thermo Fisher Scientific (2 mentions)
Cd11c pe, by Thermo Fisher Scientific (2 mentions)
Ifn γ fitc, by Thermo Fisher Scientific (2 mentions)
Facscalibur, by BD (2 mentions)
Cd25 apc, by Thermo Fisher Scientific (2 mentions)
24 protocols using il 17 pe
1
Ex vivo Analysis of T-cell Phenotype
2
Phenotypic Analysis of T Cells
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CD45-RO+ frequencies were assessed directly ex vivo by surface staining at 4 o C in PBS with antiCD45RO-FITC, CD3-PE and CD4-APC (all from BD Biosciences). For post-stimulation cultures, dead cells were labelled with near-IR LIVE/DEAD fixable dead cell stain (Molecular Probes, Life Technologies) before fixation. For analysis of regulatory markers: CTLA-4, Foxp3 and CD25, cells were fixed, permeabilised and stained with ebioscience/Thermofisher Foxp3 staining buffers according to the manufacturer's instructions. For analysis of cytokine expression, PMA/ionomycinrestimulated cells were fixed with 3% paraformaldehyde in PBS for 12 minutes followed by a 5-minute wash with PBS under centrifugation. Fixed cells were permeabilised with 0.1% saponin (Acros Organics) prepared in PBS and stained with IL-17-PE, IFNγ-e450, IL-21-APC, CD3-PERCP, CD4-FITC. For all studies cells were acquired on a Dako Cyan flow cytometer (Dako Cytomation) and data analysed using FlowJo software (Tree Star version 8.8.6). All antibodies were purchased from ebioscience/Thermofisher or BD Biosciences and expression quantified relative to the appropriate isotype control.
3
FACS Analysis of Myeloid-Derived Suppressor Cells
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Cells were stained with specific antibodies or control isotypes using conventional protocols. The following anti-mouse antibodies were used for FACS analysis of MDSCs: CD11b/PECy5, Gr1/PE, Gr1/APC, CD62L/FITC, F4/80/FITC, CD11c/PE, and MHC CI OVA peptide/Biotin from eBiosciences (San Diego, CA); as well as CD124/PE, CD40/FITC, CD86/FITC, IA-IE/FITC, Ly6C/FITC and Ly6G/PE that were purchased from BD Biosciences (San Jose, CA). Lymphocytes characterization was performed with the next specific antibodies: CD8/PE and CD8/Biotin, CD69/PECy5, CD62L/FITC, CD247/PE, CD4/PE, CD4/PECy5, CD25/PECy5, Foxp3/Biotin, IFN-γ/FITC, IL-4/PE and IL-17/PE, all from eBiosciences. FITC-conjugated Streptavidin was purchased also from eBiosciences. In all cases, cells were previously incubated with anti-mouse FcγR 2.4G2 ascites (HB-197; ATCC) to reduce the non-specific binding. For intracellular staining, cells were fixed and permeabilized, according to the manufacturer’s protocol, with either Foxp3 Staining Buffer Set or IC Fixation and 10X Permeabilization Buffers from eBiosciences. Cells were acquired using both FACScan (BD Biosciences) and Gallios (Beckman Coulter, Miami, FL) flow cytometers and analyzed with Kaluza 1.2 (Beckman Coulter) and FlowJo 5.7.2 (Tree Star Inc., USA) softwares.
4
Comprehensive Immune Cell Phenotyping
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The following antibodies were used in this study: CD3 AF700, CD4 BV421, CD4 FITC, CD6 APC, CD10 BV605, CD19 APC-Cy7, CD21 PE-Cy7, CD25 PerCP-Cy5.5, CD27 BV421, CD28 PerCP-Cy5.5, CD38 PerCp-Cy5.5, CD45RA APC-Cy7, TCR αβ PerCP-Cy5.5, IL-2 PE, LAT PE, NTAL/LAB APC, and biotin anti–human TCR-Vd2 (BioLegend); TCR-Vd1 APC (Miltenyi Biotec); CD8 APC, CD8 Pacific blue, CD21 PE-Cy7, CD31 PE, CD56 APC, CD69 FITC, CD107a PE, CD127 Alexa Fluor 647, IFN-γ FITC, TCR γδ PE, IgG Alexa Fluor 700, IκBα PE, ERK1/2(pT202/pY204) AF647, and ZAP70(pY319)/SYK(pY352) APC (BD); IgD FITC and IgA PE (SouthernBiotech); CD3 PE-Cy7, CD4 PE-Cy7, CD8 PE, CD16 FITC, CD45RA FITC, CD45 Pacific blue, Vα24 PE, and Vβ11 FITC (Beckman Coulter); CCR7 PE (R&D Systems); Bruton tyrosine kinase/ITK(pY551/pY511) PE, IL-17 PE, IL-4 APC, and ICOS PE (eBioscience); IgM Alexa Fluor 647 (Jackson ImmunoResearch Laboratories, Inc.); and PLCγ1(pY783) and goat anti–rabbit AF647 (Cell Signaling Technology). For immunohistochemistry, CD21 (Dako), CD20 (Invitrogen), CD3 (Cell Marque), CD4 and CD8 (Spring Bioscience), and Bcl-6 (Leica Biosystems) were used. For immunoblotting LAT (sc-7948; Santa Cruz Biotechnology, Inc.), FLAG tag (AHP1074; AbD Serotec), actin (sc-1616; Santa Cruz Biotechnology, Inc.), PLCγ1 (pY783; no. 2821; Cell Signaling Technology), and ZAP70 (pY319; no. 2701; Cell Signaling Technology) were used.
5
Comprehensive Immunophenotyping of T and B Cells
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Phenotypic analyses of T cells and B cells were performed with anti-human monoclonal antibodies (mAbs): anti-human CD3-PerCP, CD4-FITC, CD19-PerCP and CD21-APC were from BD Biosciences (San Jose, CA, USA). CXCR5-APC, ICOS-PE, PD-1-PE, IFN-γ-PE, IL-4-PE, IL-17-PE, IL-21-PE, IL-22-PE, CD27-FITC, CD86-PE, CD95-PE, CD25-APC, and Foxp3-PE antibodies were obtained from eBiosciences (San Diego, CA, USA). Cells were analyzed by flow cytometry (BD FACSCalibur, San Jose, CA) and data was analyzed by FlowJo software (Tree Star, San Carlos, CA).
6
Comprehensive Antibody Characterization Protocol
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Anti-SSEA4, active β-catenin and total β-catenin monoclonal antibodies were purchased from Millipore (Billerica, MA, USA). Anti-Sca1-PE, CD34-PE, CD45-PE, CD73-PE, CD4-PerCP, CD8-FITC, CD25-APC, CD3ε and CD28 antibodies were purchased from BD Biosciences (San Jose, CA, USA). Anti-CD105-PE, CD178(FASL)-PE, Foxp3-PE, IL17-PE, and IFNγ-APC antibodies were purchased from eBioscience (San Diego, CA, USA). Anti-TERT, FASL and total β-catenin (ChIP grade) polyclonal antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-BRG1 antibody was purchased from Cell Signaling (Danvers, MA, USA). Anti-β-Actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA).
7
Intracellular Cytokine Staining of Stimulated Immune Cells
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PBMCs and lymph node, colon, rectum, and jejnum cells were stimulated as previously described31 and stained with the following surface antigen antibodies from BD biosciences unless otherwise stated: CD45 PerCP (D058-1283), CD3 PE CF594 (SP34-2), CD4 Brilliant Violet 605 (Biolegend, OKT4), CD8 APC-H7 (SK1), CD14 Brilliant Violet 786 (Biolegend, M5E2). Following surface staining, stimulated cells were permeabilized using Cytofix/Cytoperm (BD biosciences), and stained using IL-17 PE (eBioscience, ebio64CAP17). T cells were defined as live/CD45+/CD20−/CD14−/CD3+ and CD4+ T cells were further delineated to assess IL-17 production.
Stained samples were fixed in 1% paraformaldehyde and collected on an LSR II (BD Biosciences, La Jolla, California). Analysis was performed in FlowJo (version 9.7.6, Treestar Inc., Ashland, Oregon).
Stained samples were fixed in 1% paraformaldehyde and collected on an LSR II (BD Biosciences, La Jolla, California). Analysis was performed in FlowJo (version 9.7.6, Treestar Inc., Ashland, Oregon).
8
Multicolor Flow Cytometry Immunophenotyping
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Mononuclear cells were immunostained with various combinations of the following fluorescence-conjugated antibodies: CD25-APC (eBioscience, San Diego, CA, USA), CD4-Paciffic blue (eBioscience), Foxp3-PE (eBioscience), H-2d-percp-cy5.5 (eBioscience), IL-17-PE (eBioscience), Foxp3-APC (eBioscience), IFN-γ-APC (eBioscience), IL-4-PE (BD PharMingen), IL-17-FITC (eBioscience), H-2b-FITC (eBioscience), IL-6-PE (Biolegend), and TNF-α-APC (BD). Before intracellular cytokine staining, cells were stimulated in culture medium containing phorbol myristate acetate (25 ng/mL; Sigma-Aldrich), ionomycin (250 ng/mL; Sigma-Aldrich), or monensin (GolgiStop, 1 μL/mL; BD PharMingen) in a 5% CO2, 37°C incubator for 4 h. Intracellular staining was performed using an intracellular staining kit (eBioscience) according to the manufacturer’s protocol. Flow cytometry was performed on a FACSCalibur flow cytometer (BD PharMingen) with the FlowJo software (TreeStar, Ashland, OR, USA).
9
Intracellular Cytokine Profiling of CD4+ T Cells
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For assessment of intracellular cytokine expression in CD4+ T cells, the lymphocytes were incubated with 10 μg/mL brefeldin A (Sigma-Aldrich, St. Louis, MO, USA). After 6 hours, cells were harvested and stained with anti-CD4-FITC (eBioscience, Inc., San Diego, CA, USA) for 30 min at room temperature, followed by fixation and permeabilization using the Cytofix/Cytoperm Kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions. The intracellular cytokine staining was performed using the following monoclonal antibodies: IFN-γ-PE, IL-4-PEcy7, IL-17-PE, and Foxp3-PEcy7 (eBioscience, Inc., San Diego, CA, USA). Stained cells were analyzed by a cube-8 flow cytometer (Sysmex/Partec, Münster, Germany). Gates were applied to define the populations of interest, and the analysis was carried out using FCS Express 4 (De Novo Software, CA, USA). Isotype controls were used as negative controls.
10
Macrophage-Derived Soluble Factors Modulate T Cell Activation
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The RAW 264.7 cells with a density of 2*105/well were cultured in 6-well plates, and after cell adhesion the medium was replaced with CS ionic solution in different dilutions (1/8, 1/16, 1/32, 1/64, and 1/128). The cell culture supernatant was collected after 72 h, and labeled as CM-control, CM-1/8CS, CM-1/6CS, CM-1/32CS, CM-1/64CS and CM-1/128CS. The complete medium was used as a control.
The naïve T cells were sorted from mouse monocytes by using mouse CD4 (L3T4) magnetic bead antibody, which was close to 100% purity (Fig. S2 ). CD28 mouse antibody (BioCell) was added into 6-well plates and incubated for 2 h. Then, the solution was removed and naïve T cells were added into the well, followed by the addition of CS ionic solution with different dilutions (1/8, 1/16, 1/32, 1/64, and 1/128), or supernatant of macrophage cultured with CS ionic solutions (CM-control, CM-1/8CS, CM-1/16CS, CM1/32CS, CM-1/64CS, CM-1/128CS).
Meanwhile, naïve T cells were also activated by adding CD3 mouse antibody (BioCell), and collected after 48 h stimulation. Then, surface staining with anti-mouse CD4-FITC was performed. After fixation and membrane rupture, cells were intracellularly stained with anti-mouse IFNγ-APC, IL17-PE, or mouse FOX-P3-APC (eBioscience, USA), and the stained cells were analyzed by flow cytometry.
The naïve T cells were sorted from mouse monocytes by using mouse CD4 (L3T4) magnetic bead antibody, which was close to 100% purity (
Meanwhile, naïve T cells were also activated by adding CD3 mouse antibody (BioCell), and collected after 48 h stimulation. Then, surface staining with anti-mouse CD4-FITC was performed. After fixation and membrane rupture, cells were intracellularly stained with anti-mouse IFNγ-APC, IL17-PE, or mouse FOX-P3-APC (eBioscience, USA), and the stained cells were analyzed by flow cytometry.
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