For assessment of cytokine production by intracellular staining, cells were cultured in RPMI (Gibco) containing 10% FBS (Gemini Bio-Products), 50 ng mL−1 PMA, and 500 ng mL−1 Ionomycin in U-bottom 96-well plates (Corning) for 1 hr at 37°C, followed by addition of 2 μM monensin (eBioscience) for another 4 hr at 37°C. Cells were then permeabilized using the Foxp3 Staining Buffer Set (eBioscience), and stained for intracellular cytokines at 4°C overnight. Fluorochrome-labeled monoclonal antibodies (clones denoted in parenthesis) against IFN-γ (XMG1.2), IL-4 (11B11), and IL-10 (JES5–16E3) were purchased from BD Biosciences, IL-17 (TC11–18H10.1) was purchased from BioLegend, and IL-21 (mhalx21) from eBioscience. All antibodies were used at a dilution of 1:100. Flow cytometry data was acquired on an LSRFortessa or LSRFortessa X-20 (BD Biosciences), or on an Aurora spectral cytometer running SpectroFlo 2.2.0.4 (Cytek Biosciences), and analyzed using FlowJo software (Tree Star).
Il 17 tc11 18h10.1
Manufactured by BioLegend
IL-17 (TC11–18H10.1) is a monoclonal antibody that recognizes the IL-17 protein. It can be used for various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa x 20, by BD (1 mentions)
96 well u bottom plate, by Corning (1 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Fetal bovine serum (fbs), by Gemini Bio (1 mentions)
Monensin, by Thermo Fisher Scientific (1 mentions)
Jes5 16e3, by BD (1 mentions)
Fixation permeabilization solution kit, by BD (1 mentions)
Na ve cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Anti il 4 and anti ifn γ neutralizing antibodies, by BioLegend (1 mentions)
Anti cd3 145 2c11, by BD (1 mentions)
Anti cd28 37.51, by BD (1 mentions)
Mil 6, by R&D Systems (1 mentions)
Golgiplug, by BD (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (1 mentions)
Lsr fortessa, by Tree Star (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Facscanto 2, by Tree Star (1 mentions)
3 protocols using il 17 tc11 18h10.1
1
Intracellular Cytokine Staining Protocol
2
Naive CD4+ T Cell Polarization
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Naïve CD4+ T cells were purified from spleens by depleting CD4-negative cells, followed by enrichment of CD62L+ cells using Naïve CD4+ T cell Isolation Kit (Miltenyi Biotec) according to the manufacture’s protocol. 1.0 × 105 cells were primed with plate-bound 2 μg/ml anti-CD3 (145-2C11; BD Pharmingen) and 2 μg/ml anti-CD28 (37.51; BD Pharmingen) in 100 μl DMEM supplemented with 10% FCS in round-bottomed 96-well plates at 37°C for 3 d in the presence of 10 μg/ml anti-IL-4 and anti-IFN-γ neutralizing antibodies (BioLegend), 50 ng/ml mIL-6 (R&D) and 1 ng/ml mouse Transforming Growth Factor-β (R&D). At day 3, 100 μl of fresh medium with the above reagents were added. At day 5, the cells were stimulated with PMA/ionomycin and fixed and permeabilized with the Fixation/Permeabilization Solution kit (BD Biosciences) and were stained for IL-17 (TC11-18H10.1, BioLegend) according to the manufacture’s protocol.
3
Profiling Cytokine Responses in Lung Cells
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Freshly isolated lung cells were stimulated for 6 hours with UV-inactivated influenza X31 (20 TCID 50 /mL), Der p 1 (0.5 mg/mL), and LPS (0.5 mg/mL) or with phorbol 12-myristate 13-acetate (10 ng/mL; Sigma) and ionomycin (500 nmol/L; Merck, Whitehouse Station, NJ) in the presence of GolgiPlug (BD) for the final 4 hours of culture. A Cytofix/Cytoperm kit (BD) was used for cell permeabilization, staining, and subsequent washing. The following antibodies (clone ID in parentheses) were used at concentrations recommended by the manufacturers: IL-21 (FFA21), IL-13 (eBio13A), IFN-g (XMG1.2) (all from eBioscience), IL-17 (TC11-18H10.1) (from BioLegend), and IL-6 (MP5-20F3; from BD Biosciences). Data were acquired on an LSRFortessa or FACSCanto II instrument and were analyzed with FlowJo software (TreeStar, Ashland, Ore).
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