Il 17a

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, China, Japan, Lao People's Democratic Republic, France

IL-17A is a cytokine that plays a key role in the inflammatory response. It is produced by a variety of immune cells and is involved in the regulation of immune responses. The core function of IL-17A is to induce the production of other cytokines and chemokines, which can lead to the recruitment and activation of neutrophils and other inflammatory cells.

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IL-17A (BMS635; Anti-IL-17A-PE IL-17A (clone eBio64DEC17) Anti-IL-17A-APC IL-17A Mouse Uncoated ELISA Kit IL-17A-Percp (17B7) PerCP-Cy5.5 anti-mouse IL-17A (eBio17B7, Mouse IL-17A ELISA Kit IL-17A PE-Cyanine7 IL-17A (clone eBio17B7,

306 protocols using il 17a

Multiparameter Flow Cytometry Staining

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The cell staining procedure used in this study was previously described by Zhan and colleagues with slight modification (19 (link)). Intracellular cytokines were stained using the intracellular fixation/permeabilization buffer set (eBioscience, CA, United States). Flow cytometric analysis was performed on a FACS Fortessa flow cytometer (BD, NJ, New York, United States). Data were analyzed using FlowJo software (version 10.0.7; Tree Star).
Antibodies were purchased from the following companies. Anti-human: CD3 (BD Biosciences, briefly called BD), CD4 (BD), CD19 (BD), CD14 (BD), TLT2 (BioLegend), IFN-γ (eBioscience), IL-4 (eBioscience), IL-17A (eBioscience), IL-6 (BD), Fixable Viability Dye (eBioscience). Anti-mouse: CD3 (BD), CD4 (BD), F4/80 (eBioscience), CD11b (BD), IFN-γ (eBioscience), IL-4 (eBioscience), IL-17A (eBioscience), IL-6 (BioLegend), and TLT2 (BioLegend).

Li J., Cao C., Xiang Y., Hong Z., He D., Zhong H., Liu Y., Wu Y., Zheng X., Yin H., Zhou J., Xie H, & Huang X. (2020). TLT2 Suppresses Th1 Response by Promoting IL-6 Production in Monocyte Through JAK/STAT3 Signal Pathway in Tuberculosis. Frontiers in Immunology, 11, 2031.

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ELISpot Assay for SARS-CoV-2-Specific Immune Responses

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ELISpot plates (MilliporeSigma) were coated with either IFNγ (BD Biosciences, #51–2525KZ), IL-17A (Invitrogen, #88–7371–88), or IL-5 (BD Biosciences, #51–1805KZ) capture antibodies at a 1:200 dilution in Dulbecco’s PBS (DPBS; Gibco). After overnight incubation at 4 ^oC, plates were washed and then blocked with complete RPMI (cRPMI) medium for at least 2 h. Splenocytes harvested as described above were plated at 2 × 10⁵ cells per well. A subset of each sample was stimulated with PepMix SARS-CoV-2 (JPT Peptide Technologies, #PM-WCPV-S-1) at a final concentration of 1 µg/mL. Plates were then incubated at 37 °C and 5% CO2 for 48 h. After a wash with PBS with 0.1% Tween 20, 100 µL of detection antibody (IFNγ, BD Biosciences, #51–1818KA; IL-17A, Invitrogen, #88–7371–88; and IL-5, BD Biosciences, #51–1806KZ) was added at a 1:250 dilution in ELISpot diluent (eBioscience) overnight at 4 °C. Plates were washed and developed with Vector NovaRED Substrate Peroxidase (Vector Laboratories, Burlingame, CA; #SK-4800) for 15 min. The reaction was stopped by washing the plates with deionized water, and plates were left to dry in the dark. Spots were counted and data were analyzed using ImmunoSpot 7 software (Cellular Technology Limited, Cleveland, OH).

Voigt E.A., Gerhardt A., Hanson D., Jennewein M.F., Battisti P., Reed S., Singh J., Mohamath R., Bakken J., Beaver S., Press C., Soon-Shiong P., Paddon C.J., Fox C.B, & Casper C. (2022). A self-amplifying RNA vaccine against COVID-19 with long-term room-temperature stability. NPJ Vaccines, 7, 136.

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SARS-CoV-2 Specific T-cell ELISpot Assay

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ELISpot plates (MilliporeSigma) were coated with either IFNγ (BD Biosciences, #51-2525KZ), IL-17A (Invitrogen, #88-7371-88), or IL-5 (BD Biosciences, #51-1805KZ) capture antibodies at a 1:200 dilution in ELISpot coating buffer (eBioscience, Waltham, MA). After an overnight incubation at 4 o C, plates were washed and then blocked with complete RPMI (cRPMI) medium for at least 2 hours. Splenocytes harvested as described above were plated at 2 x 10 5 cells per well. A subset of each sample was stimulated with PepMix SARS-CoV-2 (JPT Peptide Technologies, #PM-WCPV-S-1) at a final concentration of 1 µg/mL. Plates were then incubated at 37°C and 5% CO2 for 48 hours. After a wash with PBS with 0.1% Tween 20, 100 µL of detection antibody (IFNγ, BD Biosciences, #51-1818KA; IL-17A, Invitrogen, #88-7371-88; and IL-5, BD Biosciences, #51-1806KZ) was added at a 1:250 dilution in ELISpot diluent (eBioscience) overnight at 4 o C. Plates were washed and developed with Vector NovaRED Substrate Peroxidase (Vector Laboratories, Burlingame, CA; #SK-4800) for 15 minutes. The reaction was stopped by washing the plates with deionized water, and plates were left to dry in the dark. Spots were counted and data were analyzed using ImmunoSpot software (Cellular Technology Limited, Cleveland, OH).

Voigt E.A., Gerhardt A., Hanson D., Battisti P., Reed S., Singh J., Mohamath R., Jennewein M.F., Bakken J., Beaver S., Press C., Soon‐Shiong P., Paddon C.J., Fox C.B., & Casper C. (2022). A self-amplifying RNA vaccine against COVID-19 with long-term room-temperature stability.

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Th17 Cell Differentiation and Renal Cell Response

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We followed the methods reported by Xu et al. for cell culture [21 (link)]. A 6-well assay plate precoated with anti-CD3 and anti-CD28 Ab (BD Pharmingen) was incubated at 37 °C for 4 h. Splenocytes (1 × 10⁶ cells/well) were obtained from the spleens of 3 to 4-weeks-old mice and cultured for 3 d in 6-well plates in a medium containing TGF-β (2 ng/mL; Peprotech), IL-6 (20 ng/mL; Peprotech), anti-IFN-γ (10 μg/mL; eBioscience), and anti-IL-4 (10 μg/mL; eBioscience). These cells were treated with DMSO or Am80 (1 or 10 nmol/L) for 72 h and then collected for the Th17 cell differentiation test using flow cytometry, while the cell supernatants were used for the detection of IL-17A using enzyme-linked immunosorbent assay (ELISA).
The rat renal tubular epithelial cell line, NRK-52E, was purchased from GuangZhou Jennio Biotech and cultured in F12/DMEM (Hyclone) with 10% fetal calf serum (GIBCO). After pre-incubation in F12/DMEM without serum for 8 h, the NRK-52E cells were treated with PBS, TGF-β (10 ng/mL; Peprotech), IL-17A (10 ng/mL; Peprotech), or TGF-β (10 ng/mL) + IL-17A (10 ng/mL) for 48 h.

Li L., Luo R., Yang Y., Cheng Y., Ge S, & Xu G. (2020). Tamibarotene inhibit the accumulation of fibrocyte and alleviate renal fibrosis by IL-17A. Renal Failure, 42(1), 1173-1183.

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Cytokine Stimulation of WPMY-1 Cells

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WPMY-1 cell lines were obtained from the Cell Culture Center of the Chinese Academy of Medical Sciences (Shanghai, China). Cells were cultured in DMEM high glucose medium supplemented with 5% fetal bovine serum and were maintained at 37°C with 5% CO₂. Cells were stimulated with IFN-γ (20 ng/ml), IL-17A (20 ng/ml), and both IFN-γ (20 ng/ml) and IL-17A (20 ng/ml) (PeproTech, Rocky Hill, USA) for 48 h; cells were collected for RT-qPCR; and supernatants were collected for ELISA.

Hua X., Ge S., Zhang M., Mo F., Zhang L., Zhang J., Yang C., Tai S., Chen X., Zhang L, & Liang C. (2021). Pathogenic Roles of CXCL10 in Experimental Autoimmune Prostatitis by Modulating Macrophage Chemotaxis and Cytokine Secretion. Frontiers in Immunology, 12, 706027.

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Cytokine Stimulation of Splenocytes

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Spleens were aseptically removed from naïve WT, Il21r^-/-, STAT3^flox/flox or CD4^stat3-/- mice and cells were mechanically disrupted through a 100 μm cell strainer (BD Biosciences, San Jose, CA) using the plunger of a 6 ml syringe. RBCs were lysed in ACK lysing buffer (Lonza, Walkersville, MD), and the remaining cells were washed twice with ice-cold PBS. A total of 5×10⁶ splenocytes were cultured in duplicate in 1 ml of RPMI 1640 (Gibco) supplemented with 10% FBS and 100 μg/ml penicillin/streptomycin (all from Gibco). Cells were allowed to rest for 2 additional hours at 37°C and subsequently were stimulated in the presence of recombinant murine IFN-γ, IL-17A, IL-21 and IL-22 (20 ng/ml; Peprotech, Rocky Hill, New Jersey, USA) alone or in combination (IFN-γ+IL-17A; IFN-γ+IL-21; IFN-γ+IL-22; 20 ng/ml each). Cells receiving no cytokine treatments were used as controls. Cells were harvested at different intervals following the addition of cytokines and were stained with indicated antibodies.

Solaymani-Mohammadi S, & Berzofsky J.A. (2019). Interleukin 21 collaborates with interferon-γ for the optimal expression of interferon-stimulated genes and enhances protection against enteric microbial infection. PLoS Pathogens, 15(2), e1007614.

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Cytokine-Induced Response in Renal Epithelial Cells

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Primary renal tubular epithelial cells (RTEC) from C57BL/6J mice (Cell Biologics, Chicago, IL) were cultured as per manufacturer’s instructions. RTEC (1x10⁶ cells/well) were treated with IL-17A (50 or 200 ng/ml) or TNFα (5 ng/ml) or IL-17C (50 or 200 ng/ml) or IL-17F (50 or 200 ng/ml) and IL-17 and TNFα in combination for 24 h. Recombinant murine IL-17A, IL-17C, IL-17F and TNFα were purchased from Peprotech (Rocky Hill, NJ).
Bone marrow derived macrophages (BMDM) from C57BL/6J mice were cultured for 7 days in the presence of L929 supernatants. BMDM and RTEC (1x10⁶ cells/well) were treated with bradykinin (R&D Biosystems, Minneapolis MN) or left untreated for 24 h. LPS (Sigma Aldrich, St Louis, MO) was used as positive control. Supernatants were subjected to analyses using commercially available IL-6, IL-1β and TNFα ELISA kits (Ebiosciences, Dallas TX. Nitrite concentrations were measured by tri-iodide based reductive chemiluminescence as previously described [57 (link)]. Briefly, samples were injected into tri-iodine to reduce nitrite to NO gas that was detected by a Nitric Oxide Analyzer (Sievers, GE).

Ramani K., Garg A.V., Jawale C.V., Conti H.R., Whibley N., Jackson E.K., Shiva S.S., Horne W., Kolls J.K., Gaffen S.L, & Biswas P.S. (2016). The Kallikrein-Kinin System: A Novel Mediator of IL-17-Driven Anti-Candida Immunity in the Kidney. PLoS Pathogens, 12(11), e1005952.

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Macrophage-T cell Coculture Protocol

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For macrophage and T cell coculture experiments, bone marrow–derived monocytes (BMDMs) were isolated and differentiated into macrophage as previously described. At day 0, BMDMs were seeded at 60,000 cells/cm² and incubated at 37°C for 7 days in medium supplemented with macrophage colony-stimulating factor (100 ng/ml) (PeproTech) into a 12-well insert of a transwell plate (Corning). Naïve CD4⁺ T cells were isolated from murine lymph nodes at day 4 using a magnetic bead negative selection kit (Miltenyi). Then, T cells were seeded at 1 million cells/ml in 2 ml into six-well plates (Corning) in naïve condition supplemented with IL-2 (100 ng/ml) (PeproTech) or skewed toward the T_H17 phenotype using a commercially available kit (R&D Systems). At day 7, differentiated macrophages were exposed to naïve and T_H17 T cells at 250,000 cells/cm² or IL-17A, IL-17F (PeproTech, 100 ng/ml), and control medium for 72 hours.
Fibroblasts were isolated using previously reported methods (45 ), seeded to T-75 tissue culture plates (Corning), and passaged once before reseeding at 50,000 cells/cm² for 24 hours and exposed to IL-36γ (R&D Systems) or IL-17A (PeproTech) at 100 ng/ml for 24 hours. Cells were lysed using RLT plus buffer (Qiagen) reagent for quantitative reverse transcription polymerase chain reaction (qRT-PCR).

Sommerfeld S.D., Cherry C., Schwab R.M., Chung L., Maestas DR J.r., Laffont P., Stein J.E., Tam A., Ganguly S., Housseau F., Taube J.M., Pardoll D.M., Cahan P, & Elisseeff J.H. (2019). Interleukin-36γ–producing macrophages drive IL-17–mediated fibrosis. Science immunology, 4(40), eaax4783.

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Cytokine Levels in Bronchoalveolar Lavage Fluid

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Cytokine levels in the BALF were measured using the following enzyme-linked immunosorbent assay (ELISA) kits: interleukin (IL)-8, IL-12p70, IL-17A, IL-22, interferon (IFN)-γ (eBioscience Inc., San Diego, CA, USA), IL-6 (R&D Systems, Minneapolis, MN, USA), and IL-18 (MBL, Nagoya, Japan). The sensitivity of these assays was 4 pg/mL for IFN-γ, 0.1 pg/mL for IL-12, 0.7 pg/mL for IL-6, 2 pg/mL for IL-8, 8 pg/mL for IL-22, 12.5 pg/mL for IL-18, and 30 pg/mL for IL-17A. Undetectable values were assigned an arbitrary value of half the sensitivity limit.

Inomata T., Konno S., Nagai K., Suzuki M, & Nishimura M. (2018). Neutrophil predominance in bronchoalveolar lavage fluid is associated with disease severity and progression of HRCT findings in pulmonary Mycobacterium avium infection. PLoS ONE, 13(2), e0190189.

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Quantifying Inflammatory Mediators in BALF

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The mouse ELISA kits were used to measure the levels of HMGB1 (Shino-TEST Co, Tokyo, Japan), TGF-β, TNF-α, IL-1β, IL-17A, and IL-23 (p19) (eBioscience) in BALF, serum and co-cultures according to the manufacturer's instructions.

Zhou M., Fang H., Du M., Li C., Tang R., Liu H., Gao Z., Ji Z., Ke B, & Chen X.L. (2019). The Modulation of Regulatory T Cells via HMGB1/PTEN/β-Catenin Axis in LPS Induced Acute Lung Injury. Frontiers in Immunology, 10, 1612.

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