Concentrations of inflammatory cytokines in colon tissue were measured by commercial ELISA kit according to the manufacturer's instructions. Mouse IL-1β, IL-6, interferon (IFN)- γ, and tumor necrosis factor (TNF)-α levels were determined using Duoset ELISA (R&D Systems, Minneapolis, MN, USA) and IL-17A levels was measured with IL-17A ELISA kit (Biolegend). The absorbance was measured at 450 nm with a microplate reader (Tecan, Mannedorf, Switzerland). The concentrations of colonic cytokines were normalized to the total intestinal protein concentration as determined by a bicinchoninic acid assay kit (ThermoFisher scientific, Waltham, MA, USA).
Il 17a elisa kit
Manufactured by BioLegend
Sourced in United States
The IL-17A ELISA kit is a laboratory test used to detect and quantify the levels of IL-17A, a cytokine involved in inflammatory processes, in biological samples. The kit utilizes the enzyme-linked immunosorbent assay (ELISA) technique to measure the concentration of IL-17A present in the sample.
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Lab products found in correlation
Mouse ifn γ elisa, by BioLegend (3 mentions)
Bicinchoninic acid assay kit, by Thermo Fisher Scientific (1 mentions)
Duoset elisa, by R&D Systems (1 mentions)
Microplate reader, by Tecan (1 mentions)
Ifn γ elisa kit, by BioLegend (1 mentions)
Cd4 pe cy5, by BD (1 mentions)
Ifn γ pe cy7, by BD (1 mentions)
Il 17a pe, by Thermo Fisher Scientific (1 mentions)
Dtx 880 multimode detector, by Beckman Coulter (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Lsr 2, by BD (1 mentions)
Golgiplug, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Cck 8 kit, by Dojindo Laboratories (1 mentions)
Centrifuge 5415d, by Eppendorf (1 mentions)
Interleukin 6 (il 6), by BioLegend (1 mentions)
8 protocols using il 17a elisa kit
1
Measuring Colon Cytokine Levels
2
Quantifying Murine IL-17A and Antibodies
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IL-17A levels were determined in mouse serum using a commercial murine IL-17A ELISA kit (BioLegend). Serum antibody titers to mBSA were determined by coating microtiter plates with mBSA diluted in PBS (5 µg/ml). Nonspecific binding sites were blocked with 5% (wt/vol) milk extract in PBS containing 0.05% (wt/vol) Tween-20. Sera were diluted 1/1,000 in PBS containing milk extract and Tween-20 before adding to the microplates for 2 h. Antibody levels were detected using HRP-conjugated anti–mouse IgG and tetramethylbenzidine substrate. Absorbance was measured at 450 nm.
3
IL-17A Clearance in Mice
4
Intracellular Cytokine Profiling in RA
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For intracellular cytokine staining, CD4 T cells co-cultured with monocytes for 7 days were re-stimulated 6 hours with 50ng/ml PMA (Sigma-Aldrich Corp. St. Louis, MO) and 1 ug/ml ionomycin (Sigma-Aldrich) in the presence with Golgiplug (BD Bioscience) for last 4 hours. The cells were fixed and permeablized with BD Cytofix/Cytoperm kit and stained with the following antibodies: PE-IL-17A (eBioscience), PE-Cy7-IFN-γ, and PE-Cy5-CD4 (both from BD Biosciences), followed by analysis using a BD LSRII. In some experiments, intracellular cytokine staining was performed with freshly isolated PBMC and SFMC from RA patients. The amount of IL-17A and IFN-γ in coculture supernatant was quantified by commercial ELISA kits (eBioscience for IL-17A ELISA kit and BioLegend for IFN-γ ELISA kit). The measurement of OD (Optical density) was performed using DTX 880/multimode detector (Beckman coulter, Brea, CA).
5
Splenocyte Stimulation Assay for Cytokine Production
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Splenocytes were suspended in complete media (RPMI 1640 with 10% FBS) at a concentration of 2 × 106 cells per mL. The cells were stimulated with or without 10 μg mL−1 of protein at 37 °C for 5 days. The supernatants were collected, and the levels of IFN-γ and IL-17A were determined by ELISA using mouse IFN-γ ELISA and IL-17A ELISA kits (Biolegend), respectively.
6
Measuring Cytokine Levels in Stimulated Splenocytes
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The amounts of IFN-γ and IL-17A in the cell culture supernatants were determined based on a previously described method30 (link). Spleens were aseptically removed, and cells were suspended at a concentration of 2 × 106 cells/mL in complete media (RPMI 1640 with 10% FBS). The cells were stimulated with or without 10 μg/mL of IsdB, ClfA33-213, or IC protein at 37 °C for 5 days. The supernatants were collected, and the amounts of IFN-γ and IL-17A were determined by ELISA using mouse IFN-γ ELISA and IL-17A ELISA kits (Biolegend), respectively.
7
Splenocyte Proliferation and Cytokine Analysis
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Splenocyte proliferation assay was performed using CCK-8 kits (Dojindo, Japan). Splenocytes were suspended in complete media (RPMI 1640 with 10% FBS) at a concentration of 2.5 × 106 cells per mL. The cells were stimulated with or without 10 μg mL−1 of protein at 37 °C for 3 days. The results were expressed as the proliferation index (PI), which was calculated based on the following formula: PI = OD (450 nm) for stimulated cultures/OD (450 nm) for non-stimulated cultures.
The supernatants were collected for cytokine assay and the levels of IFN-γ, IL-4 and IL-17A were determined through ELISA using mouse IFN-γ ELISA, IL-4 ELISA and IL-17A ELISA kits (Biolegend, San Diego, CA, USA), respectively.
The supernatants were collected for cytokine assay and the levels of IFN-γ, IL-4 and IL-17A were determined through ELISA using mouse IFN-γ ELISA, IL-4 ELISA and IL-17A ELISA kits (Biolegend, San Diego, CA, USA), respectively.
8
Quantifying Inflammatory Cytokine Levels
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Blood was collected from lateral tail veins of pregnant dams 3 and 48 hours after poly(I:C) or PBS injection. Fresh blood was left undisturbed at room temperature for 30 min to clot. Serum was isolated from clotted blood by centrifuging for 15 min at 1000 rpm at 4°C (Eppendorf Centrifuge 5415D) and was diluted 10 times for interleukin enzyme-linked immunosorbent assay (ELISA) tests. IL-6 (Invitrogen, #88-7064) and IL-17A ELISA kits (BioLegend, #432501) were used to test the concentration of IL-6 and IL-17A in the serum following the manufacturer’s instructions. Briefly, standard IL-6 and IL-17A provided in the kits were diluted sequentially and applied to make a standard curve. Absorbances at 450 nm (A450) were read, and the concentrations of IL-6 and IL-17A of samples were calculated according to A450 and the standard curve. The original concentration of plasma was calculated accordingly.
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