Sera were collected following centrifugation of heparinized blood (1,300 × g, 10 min). Samples were frozen at -80°C until the day of analysis using the Luminex technology (62 (link)). Levels of TNFα, IL-6, IL-1β, IL-10, IP-10, and IFNα in sera were quantified using a human cytokine magnetic bead assay (Milliplex® MAP Multiplex Assays; EMD Millipore, Billerica, MA, USA). Quantification was performed according to the manufacturer’s instructions with a sample incubation step overnight, at 4°C. Data were acquired on a Luminex® 200TM System using the Luminex xPonent® software and analyzed using the Milliplex® Analyst 5.1 software (EMD Millipore).
Luminex 200tm system
Manufactured by Merck Group
Sourced in Germany
The Luminex® 200TM System is a multiplex assay platform that utilizes xMAP® technology to perform various bioassays simultaneously. The system employs color-coded magnetic microspheres, flow cytometry, and sophisticated data analysis software to enable high-throughput analysis of multiple analytes in a single sample.
Automatically generated - may contain errors
2 protocols using luminex 200tm system
1
Multiplex Cytokine Quantification in Sera
2
Cytokine and Chemokine Quantification
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The TNF-α, IL-8, and MCP-1 levels in supernatants collected from THP-1 and HUVEC cultures were measured using commercial ELISA kits (R&D Systems, Minneapolis, MN, USA), and NF-κB activity in HUVECs was measured using the Trans AM NF-κB p65 Kit (Active Motif, Carlsbad, CA, USA). TNF-α, CCL-4/macrophage inflammatory protein (MIP)-1β, IL-6, and IL-1β levels in mouse plasma collected through cardiac puncture at the indicated time points after the high-grade CLP procedure were measured using the Mouse Magnetic Luminex Screening Assay Kit (R&D Systems). The assay was performed according to the manufacturer’s instructions. All samples and standards were assayed in duplicate using the Luminex 200TM System (Merck Millipore, Darmstadt, Germany). To measure serum TNF-α, IL-1β, IL-6, and CCL4 at the indicated time points, blood samples were collected through cardiac puncture after the LPS endotoxemia procedure, allowed to clot for 2 hours at room temperature, and then centrifuged at 2000g for 20 min at 4°C.
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