PFCs were resuspended in complete RPMI-1640 medium, stimulated with 2 μg/mL peptides plus 1 μg/mL anti-CD28, 1 μg/mL anti-CD49d mAbs, or PMA (20 ng/ml; Sigma Aldrich, Saint Louis, MO, USA) plus ionomycin (1 μg/mL; Sigma-Aldrich). PFCs were cultured in the presence of medium alone, IL-6 (30 ng/mL; Peprotech), IL-12 (5 ng/ml; eBioscience, Santiago, Chile), IL-21 (50 ng/mL; Peprotech), IL-27 (40 ng/ml; eBioscience) alone, E/C peptides, or E/C peptides plus cytokines.
Il 27
Manufactured by Thermo Fisher Scientific
Sourced in United States
IL-27 is a cytokine that belongs to the interleukin-6/IL-12 family. It is a heterodimeric protein composed of Epstein-Barr virus-induced gene 3 (EBI3) and p28 subunits. IL-27 is primarily produced by antigen-presenting cells and plays a role in the regulation of immune responses.
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Lab products found in correlation
Interleukin 6 (il 6), by Thermo Fisher Scientific (4 mentions)
Il 21, by Thermo Fisher Scientific (3 mentions)
Il 10, by R&D Systems (3 mentions)
Ifn γ, by Thermo Fisher Scientific (3 mentions)
Ionomycin, by Merck Group (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Mouse anti il 10, by BD (2 mentions)
Il 12 23p40, by BD (2 mentions)
Interleukin 2 (il 2), by R&D Systems (2 mentions)
Il 12, by R&D Systems (2 mentions)
Il 17, by BD (2 mentions)
Il 12, by Thermo Fisher Scientific (2 mentions)
Anti il 4, by Thermo Fisher Scientific (2 mentions)
Interleukin 6 (il 6), by R&D Systems (2 mentions)
Il 23, by Thermo Fisher Scientific (2 mentions)
Golgiplug, by BD (2 mentions)
Il 10, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Anti ifn γ, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ xmg1, by BioLegend (1 mentions)
25 protocols using il 27
1
Cytokine-mediated PFC activation assay
2
Cytokine Production Assessment by ELISA
3
Differentiation of Murine CD4+ T Cell Subsets
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Naïve CD4+ T (CD4+CD44low) cells from spleens of C57BL/6 mice were sorted, and activated with plate-bound 2 μg/ml anti-CD3 (145-2C11; BioLegend) and 1 μg/ml anti-CD28 (37.51; BioLegend) antibodies. The cells were treated with various cytokines or antibodies for polarization. For Th1 cell differentiation, 10 μg/ml anti-IL-4 (11B11; BioLegend) antibody and 10 ng/ml IL-12 (R&D) were used. For Th17 polarization, 20 ng/ml IL-6 (R&D), 5 ng/ml TGF-β (R&D), 10 μg/ml anti-IFN-γ (XMG1.2; BioLegend) and 10 μg/ml anti-IL-4 antibodies were used. For Tr1 cell differentiation, cells were treated with 50 ng/ml IL-27 (eBioscience), 10 μg/ml anti-IFN-γ and 10 μg/ml anti-IL-4 antibodies. For iTreg polarization, 1 ng/ml TGF-β, 4 ng/ml IL-2 (R&D), 10 μg/ml anti-IFN-γ and 10 μg/ml anti-IL-4 antibodies were used. The cells were cultured at 37 °C for 48 or 72 h, followed by flow cytometry analysis.
For detection of activation induced cell death, Naïve CD4+ T cells were activated with plate-bound 2 μg/ml anti-CD3 and 1 μg/ml anti-CD28 antibodies. 72 h later, cells were collected, and subjected to Annexin V/7-AAD staining.
For detection of activation induced cell death, Naïve CD4+ T cells were activated with plate-bound 2 μg/ml anti-CD3 and 1 μg/ml anti-CD28 antibodies. 72 h later, cells were collected, and subjected to Annexin V/7-AAD staining.
4
Cytokine Profiling by ELISA
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Blood samples (10 ml) were collected in sodium heparin tubes and plasma was collected by centrifugation at 2,600 revolutions per minute (rpm) for 10 min at 4°C and stored at −80°C. The following cytokines-IL-1α, IL-Ra, IL-1β, IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-15, IL-18, IL-21 (DuoSet R&D Systems), IL-23, IL-27, IL-33, IL-35 (e-Biosciences) and TGFβ (BioLegend) were measured using ELISA.
5
Cytokine Quantification via ELISA
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To evaluate the production of cytokines, ELISAs were performed by the Immunoassay Core of the CGIBD at UNC according to the manufacturer’s instructions with the following products: mouse anti-IL10, IL12/23p40, interferon-γ (IFNγ), and IL17 (BD Biosciences), and IL27 (eBioscience). Concentrations of cytokines were established in triplicate culture supernatants by comparison with standard curves generated using the appropriate recombinant cytokine.
6
Quantifying IL-27 Levels and T-cell Marker Expression
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Enzyme-linked immunosorbent assay (ELISA) kits were used to determine the concentrations of IL-27 (eBioscience, San Diego, USA) in MPE and serum according to manufacturer's instructions. The minimum detectable concentration of IL-27 was 9.5 pg/ml.
The expression of markers on T-cells from MPE and blood was determined by flow cytometry as described previously[16 (link)] after surface or intracellular staining with anti-human-specific Abs conjugated with FITC, PE, or APC. These human Abs included anti-CD3, -CD8, -IL-27. Intracellular staining was performed on T-cells stimulated with PMA (50 ng/ml), ionomycin (1 μmol/L), GolgiStop, and GolgiPlug for 5 h, and then stained with anti-IL-27 with PE. Flow cytometry was performed on a FACS Canto II (BD Biosciences, Franklin Lakes, NJ) and analyzed using FCS Express 4 software (De Novo Software, Los Angeles, USA).
The expression of markers on T-cells from MPE and blood was determined by flow cytometry as described previously[16 (link)] after surface or intracellular staining with anti-human-specific Abs conjugated with FITC, PE, or APC. These human Abs included anti-CD3, -CD8, -IL-27. Intracellular staining was performed on T-cells stimulated with PMA (50 ng/ml), ionomycin (1 μmol/L), GolgiStop, and GolgiPlug for 5 h, and then stained with anti-IL-27 with PE. Flow cytometry was performed on a FACS Canto II (BD Biosciences, Franklin Lakes, NJ) and analyzed using FCS Express 4 software (De Novo Software, Los Angeles, USA).
7
Investigating Immune Cell Signaling Pathways
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The following antibodies were used as follow: For flow cytometry determination, CD3, CD8, CD54 (intercellular adhesion molecule [ICAM]-1), CD106 (vascular adhesion molecule [VCAM]-1), CD11a (lymphocyte function-associated antigen-1 [LFA-1]), CD29 (integrin β1), IL-27, IFN-γ, IL-17, phosphorylated (p)-STAT1 (pY701), p-STAT1 (pS727), p-STAT2 (pY689), p-STAT3 (pS727), p-STAT3 (pY705), p-STAT4 (pY693), p-STAT5 (pY694), p-STAT6 (pY641), TCCR-WSX-1 (IL-27 receptor), IL-17 receptor (R), IFN-γR1, and Ki-67 mAbs were purchased from BD Biosciences (Franklin Lakes, USA), eBioscience (San Diego, USA), or R and D systems (Minneapolis, USA); for cytokine neutralization, IL-27, IFN-γ, IL-17, ICAM-1 and VCAM-1 mAbs were from eBioscience.
Recombinant human IL-27, IFN-γ, IL-17A were purchased from R and D systems or eBioscience. STAT1 inhibitor S14-95 and STAT3 inhibitor Galiellalactone were from Enzo Life Sciences (Farmingdale, USA). Annexin V Apoptosis Detection Kit was from eBioscience. PMA and ionomycin were from Sigma-Aldrich (St. Louis, USA), and GolgiPlug and GolgiStop were from BD Bioscience. Foxp3 fixation/permeabilization concentrate and diluent were from eBioscience. Fluorescent dye CFSE was from Invitrogen (Carlsbad, USA).
Recombinant human IL-27, IFN-γ, IL-17A were purchased from R and D systems or eBioscience. STAT1 inhibitor S14-95 and STAT3 inhibitor Galiellalactone were from Enzo Life Sciences (Farmingdale, USA). Annexin V Apoptosis Detection Kit was from eBioscience. PMA and ionomycin were from Sigma-Aldrich (St. Louis, USA), and GolgiPlug and GolgiStop were from BD Bioscience. Foxp3 fixation/permeabilization concentrate and diluent were from eBioscience. Fluorescent dye CFSE was from Invitrogen (Carlsbad, USA).
8
Isolation and in vitro differentiation of T cell subsets
9
Modulating Cellular Responses to IL-27 and TGF-β1
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The MRC-5 (human lung fibroblast-derived) cell line was purchased from the Chinese Academy of Sciences Cell Bank and cultured in minimum essential medium (MEM) containing 10% fetal bovine serum, penicillin (100 μg/mL) and streptomycin 100 (μg/mL). The cell lines were placed in a humidified atmosphere containing 5% CO2 at 37 °C. After pretreatment with PD98059 (1 μmol/L, TOCRIS, Bristol, UK), SB203580 (100 nmol/L, TOCRIS, Bristol, UK), or 3-methyladenine (20 mmol/L, 3-MA, Absin, Shanghai, China) for 2 h, MRC-5 cells were exposed to IL-27 (100 ng/mL, eBioscience, California, USA) and/or TGF-β1 (40 ng/mL, eBioscience, CA, USA) for 48 h.
10
Tumor-Infiltrating CD8+ T Cell Functional Analysis
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Tumor-infiltrating CD8 + T cells were purified from B16F10-OVA tumor cell suspensions using EasySep Mouse CD8 Positive Selection Kit II (StemCell). Cells were then sorted into PD-1 -Tim-3 -, PD-1 + Tim-3 -and PD-1 + Tim-3 + subsets using a FACS Aria IIb Cell Sorter (BD Biosciences). In vitro stimulation was performed for 72 h with plate-bound anti-CD3 (1 µg/mL, eBioscience), anti-CD28 (0.25 µg/mL, BD Biosciences) and sometimes with 200 U/mL of mouse rIL-2 (eBioscience). Cocultures: 5x10 4 sorted tumor-infiltrating CD8 + T cells were incubated in a 1:1 ratio with responder CD8 + T cells obtained from spleens from CD45.1 or CD73KO mice and stained with CFSE (Invitrogen). Cells were stimulated in vitro in the presence or absence of the ecto-ATPase inhibitor ARL67156 (Tocris). CD39 induction: CD8 + T cells magnetically purified from mice dLNs nodes were stimulated in vitro in the presence of mouse recombinant cytokines: IL-6 (20 ng/mL; eBioscience), IL-27 (10 ng/mL; eBioscience) or maintained the last 24 h in a hypoxic atmosphere (1.5% O 2 ). Human PBMCs were stimulated with T Cell Activation-Expansion Kit (Miltenyi) according to manufacturer´s indications. Human CD8 + T cells were purified using the CD8 Microbeads kit (Miltenyi). Then 2x10 5 cells were stimulated for 72 h in the presence or absence of human recombinant IL-6 (20ng/mL; ImmunoTools) and/or IL-27 (
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