Liquichip luminex 200 system

Measurements of plasma cytokine concentrations were performed using a multiplex immunoassay kit (High Sensitivity 5-Plex Mouse ProcartaPlex™ Panel; Thermo Fisher Scientific, Darmstadt, Germany) following the manufacturer's instructions. Cytokine levels were determined using the LiquiChip luminex 200 system (Qiagen, Hilden, Germany) and the Procartaplex-analyst 1.0 software (Affymetrix, eBioscience, San Diego, USA).

Blood samples from all subjects were collected in monovettes prepared with the anticoagulant ethylene-diamine-tetra-acetic acid (EDTA). Plasma samples were obtained by centrifugation and stored at − 80 °C until analyzed.
Measurements of concentrations of the adipocytokines resistin, adiponectin, leptin, interleukin (IL)-6, IL-1β, and TNF-α in plasma samples were performed using a multiplex immunoassay (Merck Millipore, Darmstadt, Germany) following the manufacturer’s protocol. In brief, standards and samples were diluted in an equal volume of assay puffer and incubated with antibody-immobilized beads at 4 °C for 16 h. After washing, a biotinylated detection antibody was added for 1 h followed by incubation with streptavidin-phycoerythrin for 30 min at room temperature. Adipocytokine levels were determined using the LiquiChip luminex 200 system (Qiagen, Hilden, Germany).

In line with previously published procedures, plasma cytokine concentrations were analyzed using a multiplex immunoassay (High Sensitivity 5-Plex Mouse ProcartaPlex™ Panel; Thermo Fisher Scientific, Darmstadt, Germany) following the manufacturer's recommendations (18 (link)). Concentrations of plasma cytokines were quantified using the LiquiChip luminex 200 system (Qiagen, Hilden, Germany) and the Procartaplex-analyst 1.0 software (Affymetrix, eBioscience, San Diego, USA).