Magneticluminex assay human premixed multi analyte kit

We applied the Magnetic Luminex Assay Human Premixed Multi-Analyte Kit (R&D Systems, Minneapolis, MN, USA) to measure the concentration of the following chemokines and cytokines (according to the manufacturer’s instructions): chemokines C-C motif chemokine ligand 2 (CCL2), C-X-C motif chemokine ligand 1 (CXCL1), CXCL9, CXCL10 and CXCL13, and the cytokines interferon (IFN)-α, IFN-γ, interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17A, tumour necrosis factor (TNF)-α, vascular endothelial growth factor A (VEGF-A), a proliferation-inducing ligand (APRIL), B-cell-activating factor belonging to the tumour necrosis factor family (BAFF), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). All data were collected with the Luminex-100 system (Luminex, Austin, TX, USA).

In the morning, venous blood samples were collected after overnight fasting using vacuum tubes, which minimizes confounding factors, such as diet and circadian rhythm. The samples were centrifuged at 1,000 g for 10 min at 4°C, and serum was collected and stored at –80°C for subsequent analysis. Specifically, serum levels of PRR14 (Jianglaibio, China) were detected using an Enzyme-linked Immunosorbent Assay (ELISA) kit, in accordance with the manufacturer's instructions. In addition, we used the Magnetic Luminex Assay-Human Premixed Multi-Analyte kit to determine the circulating levels of VCAM-1 and sCD163 according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). The minimum detectable dose of PRR14 was 2.5 ng/ml, VCAM-1 was 2,819.6 pg/ml, and sCD163 was 1,874.8 pg/ml.

IL-6 and IL-8 level were assessed using an enzyme-linked immunosorbent assay (ELISA) performed by LSI Medience Corporation (Tokyo, Japan) with a solid phase chemiluminescent ELISA kit (QuantinGlo®; ELISA, R&D Systems, MN, United States), and an IL-8 EIA kit (Invitrogen, CA, United States). The levels of other cytokines were determined by a multiplex suspension array (Genetic Lab Co., Ltd., Sapporo, Japan) using a Luminex® assay system (Luminex Corp., Austin, TX, United States) with a magnetic Luminex® assay human premixed multi-analyte kit (R&D Systems Inc., Minneapolis, MN, United States). The assay was performed according to the manufacturer’s instructions. Briefly, all samples were diluted using Calibrator Diluent RD6-52, and 50 μL of the diluted samples were used for the assay customized to detect and quantify TNF-α, IL-1β, IL-17C, IL-17E/IL25, granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemotactic protein-1 (MCP-1), thymic stromal lymphopoietin (TSLP), thymus and activation-regulated chemokine (TARC), eotaxin, and regulated on activation normal T cell expressed and secreted (RANTES). The fluorescence intensity of each sample was measured using a Luminex® 100/200TM system (Luminex Corp., Austin, TX, US), and the concentrations of each analyte were calculated using MILLIPLEX® Analyst 5.1 (Merck Millipore Corp., Burlington, MA, United States).

Leptin (RayBiotech, Peachtree Corners, GA, USA) was quantified by ELISA according to the manufacturer’s directions. The Magnetic Luminex Assay Human Premixed Multi-Analyte Kit (R&D Systems, Minneapolis, MN, USA) was applied to measure the concentration of the following chemokines and cytokines (according to the manufacturer’s instructions): adiponectin; and the cytokines interferon (IFN)-γ, IL-4, IL-16; and tumor necrosis factor (TNF)-α. All data were collected by the Luminex-100 system (Luminex, Austin, TX, USA).

Multiplexed bead‐based immunoassays (Magnetic Luminex Assay, Human Premixed Multi‐Analyte Kit, R&D Systems) were used to measure the presence of growth differentiation factor 15 (GDF15), tumor necrosis factor receptor 1 (TNFR1), FAS, and macrophage inflammatory protein 1α (MIP1α, also referred to as CCL3). All assays were performed according to the manufacturer's protocols (Schafer et al., 2020). Sensitivities and intra‐ and inter‐assay confidence values of each protein in the assays are listed in Table 1. The assays were executed according to the protocols provided by the manufacturer.
The samples were deidentified during data collection and analysis. Samples were placed such that all treatment groups were represented on each plate in order to mitigate possible variation due to difference among plates.