A549 cells into 24-well plates were split at 40 000 cells/well. After 1 day growth, cells were treated with various steroids 1 h before overnight induction of inflammation by 2 ng ml−1 TNFα according to literature [27 (link)]. Sixteen hours after treatment, cell culture supernatants were subjected to anti-IL-6 Elisa (R&D Quantikine Elisa Human IL-6 Kit) according to the manual.
Human il 6 quantikine elisa kit
Manufactured by R&D Systems
Sourced in United States, United Kingdom
The Human IL-6 Quantikine ELISA Kit is a quantitative sandwich enzyme immunoassay designed to measure human interleukin 6 (IL-6) levels in cell culture supernates, serum, and plasma. It utilizes a solid-phase antibody and an enzyme-linked polyclonal antibody specific for IL-6 to quantify the amount of IL-6 present in the sample.
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Human il 10 quantikine elisa kit, by R&D Systems (3 mentions)
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129 protocols using human il 6 quantikine elisa kit
1
Steroids Modulate Inflammation in A549 Cells
2
Quantifying Inflammatory Markers by ELISA
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Concentrations of C-reactive protein (CRP), D-dimer and interleukin-6 (Il-6) in serum samples were measured by an enzyme-linked immunosorbent assay (ELISA) using commercially available kits: Quantikine ELISA Human C-Reactive Protein/CRP kit by R&D Systems (MN, USA) for CRP, Human D-Dimer SimpleStep ELISA kit ab196269 by Abcam (Cambridge, UK) for D-dimer and Quantikine ELISA Human Il-6 kit by R&D Systems (MN, USA) for Il-6. All assays were performed according to manufacturer’s detailed instructions. The coefficients of variance for intra-assay and inter-assay were for all assays below 8 and 10%, respectively.
3
Quantifying Cytokine Profiles in Hepatitis B
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An aliquot of 1 × 106 U937- and U937-transfected cells (GV166-U937, HBe-U937) were seeded in six-well plates. After a 24-h culture, the culture supernatant was collected and BAFF in culture supernatant were analyzed by a commercially available Quantikine Human BAFF/BLyS ELISA kit (R&D Systems, Inc.), according to the manufacturer's instructions. For IL-6, IL-10, TNF-α, and APRIL detection, U937- and U937-transfected cells (GV166-U937, HBe-U937) were seeded in six-well plates as mentioned above, and induced with lipopolysaccharide (LPS) (1 μg/mL) for 24 h before culture supernatant collection. IL-6, IL-10, TNF-α, and APRIL in these culture supernatants were analyzed by commercially available ELISA kits, including Quantikine ELISA Human IL-6 kit (R&D Systems, Inc.), Quantikine ELISA Human TNF-α kit (R&D Systems, Inc.), IL-10 Human SimpleStep ELISA kit (Abcam), and APRIL Human ELISA kit (Abcam). In addition, serum BAFF, IL-6, and IL-10 were measured in samples from 16 HBe-positive and 15 HBe-negative newly diagnosed patients with hepatitis B in our hospital by using the same ELISA kit.
4
IL6 Measurement in Ganetespib-Treated Cells
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Cells were seeded in 96-well plate and treated the next day with 30 nM ganetespib or DMSO control for 24 h. After treatment, the media from the wells were collected and enzyme-linked immunosorbent assay (ELISA) was performed to measure the amount of IL6 in the media using the Quantikine ELISA Human IL6 kit (R&D Systems).
5
Measuring IL-6 Levels by ELISA
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IL-6 media concentrations were measured using the Quantikine ® ELISA Human IL-6 kit (R&D systems, Minneapolis, MN, USA, #S6050), following the manufacturer’s instructions. Briefly, cells were plated in 150 mm dishes and allowed to grow to 70–80% confluency before media was collected. The ELISA was performed in technical triplicates, with the mean ± standard error of the mean (SEM) of three biological replicates.
6
Inflammatory Biomarker Measurement Protocol
7
Measuring IFN-β and IL6 Levels
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Conditioned culture medium was collected at the indicated times and was centrifuged at 12,000 x g for 5 min at 4°C to remove cell debris. The IFN-β and IL6 concentrations in the culture medium were then measured using a human IFN-β ELISA kit (Kamakura Techno-Science, Japan) or a Quantikine Human IL6 ELISA kit (R&D Systems, Minneapolis, MN), respectively.
8
Molecular Signaling Pathway Analyses
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The Escherichia coli 0111: B4 LPS, N-hexanoyl-D-sphingosine (C6-ceramide) and zVAD-FMK were obtained from Sigma-Aldrich (St. Louis, MO, USA). DAPK1 inhibitor and ST2825 were purchased from Medchem Express (Monmouth Junction, New Jersey, USA). Recombinant GST-DAPK1 fusion protein was obtained from Millipore (Billerica, MA). Caspase-3 activity detection kit was from Bestbio (Shanghai, China). Quantikine human IL-6 ELISA kit was from R&D Systems (Minneapolis, MN). Annexin V-FITC apoptosis detection kit was ordered from Beyotime (Nanjing, China). The following antibodies with the company and concentration were used for coimmunoprecipitation (co-IP) or western-blotting analyses: anti-Pellino1 (Abcam, 1:500), anti-MyD88 (Cell Signaling Technology, 1:500), anti-caspase-8 (Cell Signaling Technology, 1:500), anti-TRIF (Cell Signaling Technology, 1:1000), anti-RIP1 (BD Biosciences, 1:2000), anti-Flag (ProteinTech group, 1:1000), anti-phospho-DAPK1 (Sigma-Aldrich, 1:1000), anti-DAPK1 (Cell Signaling Technology, 1:1000), anti-Fbxw7 (Abcam, 1:1000), anti-pSer (Santa Cruz, 1:1000), anti-Fn14 (Cell Signaling Technology, 1:2000) and anti-GAPDH (Biosynthesis, 1:3000).
9
Quantification of IL-6 and TNF-α in Cell Culture
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Cells were seeded into six-well Cell Bind plates and treated with NE. The media from each well was sampled at 24 h. Samples were centrifuged at 2100 rpm for 10 min and the cell-free culture media supernatants were stored at −80 °C until analysis was conducted using a Quantikine human IL-6 ELISA kit (Cat#: D6050, R&D System, Minneapolis, MN, USA) or human TNF-α ELISA kit (Elisakit.com, Product 0005, Melbourne, VIC, Australia).
10
Inflammatory Biomarker Measurement Protocol
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High Sensitivity C Reactive Protein (hsCRP) was measured in serum or plasma using the Cardiac C-reactive Protein (Latex) Sensitive immunoturbidimetric assay on the automated Roche/Hitachi cobas c 311 module. The lower limit of detection, as determined by the assay manufacturer, is 0.5 mg/L. Additional details of the laboratory procedures can be found here (NAHDAP, 2018b ).
Interleukin 6 (IL-6) IL-6 assays were performed following GenWay Biotech’s Standard Operating Procedure ANA015 (High Sensitivity Human IL-6 in Serum ELISA). IL-6 was measured in serum using the Quantikine Human IL-6 ELISA KIT (R&D Systems Cat# HS600B and HS600C) and Immunoassay Control Group 10 (R&D Systems Cat#QC41). Optical density was read using the Emax precision microplate reader (Molecular Devices) set to 490 nm. The assay range is 0.255–9.755 pg/mL.
Fibrinogen was measured using the Clauss fibrinogen assay, a quantitative, clot-based, functional assay. The assay measures the ability of fibrinogen to form fibrin clots after being exposed to a high concentration of purified thrombin. The assay was run on the ACL Top 300, which adds a predetermined number of units of bovine thrombin to citrated human plasma and measures the clotting kinetics turbidometrically. The assay range is 150–1000 mg/dL.
Interleukin 6 (IL-6) IL-6 assays were performed following GenWay Biotech’s Standard Operating Procedure ANA015 (High Sensitivity Human IL-6 in Serum ELISA). IL-6 was measured in serum using the Quantikine Human IL-6 ELISA KIT (R&D Systems Cat# HS600B and HS600C) and Immunoassay Control Group 10 (R&D Systems Cat#QC41). Optical density was read using the Emax precision microplate reader (Molecular Devices) set to 490 nm. The assay range is 0.255–9.755 pg/mL.
Fibrinogen was measured using the Clauss fibrinogen assay, a quantitative, clot-based, functional assay. The assay measures the ability of fibrinogen to form fibrin clots after being exposed to a high concentration of purified thrombin. The assay was run on the ACL Top 300, which adds a predetermined number of units of bovine thrombin to citrated human plasma and measures the clotting kinetics turbidometrically. The assay range is 150–1000 mg/dL.
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