Supernatants were collected at specific time points. In a blocking assay, neutralizing antibody against human-TIMP-1 or goat IgG (R&D Systems) was added into cell culture at the beginning. Quantitative detection of human IL-6 was performed with Human IL-6 ELISA MAX™ Deluxe kit (BioLegend, San Diego, CA, USA) as instructed.
Human il 6 elisa max deluxe kit
Manufactured by BioLegend
Sourced in United States
The Human IL-6 ELISA MAX Deluxe Kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of human interleukin-6 (IL-6) in cell culture supernatants, serum, and plasma.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
96 well plate, by BD (2 mentions)
Microplate spectrophotometer system, by Molecular Devices (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Il 1 beta human uncoated elisa kit, by Thermo Fisher Scientific (1 mentions)
Il 8 human uncoated elisa kit, by Thermo Fisher Scientific (1 mentions)
Synergy htx, by Agilent Technologies (1 mentions)
Lipopolysaccharides (lps), by InvivoGen (1 mentions)
6 protocols using human il 6 elisa max deluxe kit
1
Neutralizing Antibody Blockade of IL-6
2
UVB-Induced IL-6 Secretion in HaCaT Cells
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HaCaT cells were seeded in 24-well plates (120,000 cells/well) in DMEM medium supplemented with 10% FCS. The medium was changed to 3% FCS containing the different dietary compounds at the indicated concentrations. A day later, the medium was replaced with PBS, and the cells were irradiated with a UVB lamp, as described above. The PBS was replaced with medium containing 3% FCS plus the test compounds, and the medium was collected after 6 h. IL-6 in the medium was measured using a human IL-6 ELISA MAX™ Deluxe Kit (Biolegend, San Diego, CA, USA).
3
Protein and IL-6 Quantification
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Media conditioned by the samples and collected in microtubes at the end of each experiment were centrifuged (990× g, 20 min, 4 °C) and the supernatants were used for protein and IL-6 expression level quantification. Protein determination was performed in 96-well microtiter plates using the bicinchoninic acid (BCA) methodology, using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the instructions of the manufacturer. IL-6 expression levels were assessed using enzyme-linked immunosorbent assay (ELISA) using the Human IL-6 Elisa Max™ Deluxe Kit (BioLegend, San Diego, CA, USA) according to the instructions of the manufacturer and results are presented in pg/µg of protein.
4
Cytokine Secretion in HDFs
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HDFs (104/well) were seeded and grown overnight in a 96-well plate (BD Biosciences). The cells were treated for 24 h with a mixture of rhTNF-α and different concentrations of DMSO (0.1–100%) (preincubated for 1 h). The secretion level of IL-8 was assessed with the Human IL-8 Uncoated ELISA Kit (eBioscience, Inc., San Diego, CA, USA), while that of IL-6 was measured using the Human IL-6 ELISA MAX Deluxe Kit (BioLegend, San Diego, CA, USA). The absorbance in the plate wells was read on a microplate spectrophotometer system (Molecular Devices).
5
Quantifying IL-6 in NMO-IgG Treated Cells
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After incubating A4 and A with NMO-IgG or control IgG for 24 hours, IL-6 concentration was measured in triplicate samples using human IL-6 ELISA Max Deluxe kit, per manufacturer's protocol (BioLegend, San Diego, CA).
The Cleveland Clinic Institutional Review Board approved study protocols, and signed informed consents were obtained from blood donors. We recruited healthy volunteers, aged 20–50 years, not experiencing systemic infection, and excluded individuals taking nonsteroidal anti-inflammatory drugs. Peripheral blood mononuclear cells (PBMC) were isolated with lymphocyte separation medium (Mediatech, Manassas, VA). For transmigration assays, performed within 2 hours of phlebotomy, PBMC were resuspended at 10 × 106 cells in 30 mL TEM buffer (RPMI 1640 without phenol red + 1% bovine serum albumin + 25 mM HEPES) and stained with Calcein AM (Life Technologies) before perfusion into the chamber.
The Cleveland Clinic Institutional Review Board approved study protocols, and signed informed consents were obtained from blood donors. We recruited healthy volunteers, aged 20–50 years, not experiencing systemic infection, and excluded individuals taking nonsteroidal anti-inflammatory drugs. Peripheral blood mononuclear cells (PBMC) were isolated with lymphocyte separation medium (Mediatech, Manassas, VA). For transmigration assays, performed within 2 hours of phlebotomy, PBMC were resuspended at 10 × 106 cells in 30 mL TEM buffer (RPMI 1640 without phenol red + 1% bovine serum albumin + 25 mM HEPES) and stained with Calcein AM (Life Technologies) before perfusion into the chamber.
6
Quantifying Macrophage Cytokine Responses
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THP-1 derived macrophage cells (105/well), HDFs (104/well), and 293/hTLR5 cells (3 × 104/well) were seeded and grown overnight in 96-well plates (BD Biosciences). After 24 h of treatment, TNF-α production was assessed by means of the Human TNF Alpha Uncoated ELISA Kit (eBioscience, San Diego, CA, USA); IL-6 by the Human IL-6 ELISA MAX Deluxe Kit (BioLegend, San Diego, CA, USA); IL-8 by the Human IL-8 Uncoated ELISA Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). For NLRC4 activation, THP-1 macrophages were primed with LPS (InvivoGen, San Diego, CA, USA; 1 μg/mL) for 4 h, followed by transfection of FLA-AA and FLA-ST via Lipofectamine 2000 for 2 h. The culture supernatant was collected to assess the secreted amount of IL-1β with the IL-1 beta Human Uncoated ELISA Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The plates were then analyzed on a Synergy HTX multi-mode reader (Bio-Tek, Winooski, VT, USA) at respective wavelengths.
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