Bioplex multiplex human cytokine assay

We collected blood samples for serum IL-13 determination at T0 and T1. IL-13 circulating levels were determined by using the Bioplex Multiplex human cytokine assay (Bio-Rad, Hercules, CA, United States) according to the manufacturer’s instructions. IL-13 levels in EAT, mature adipocytes and stromal vascular fraction cells secretomes were determined as described in Supplementary Material.

AGS cells were split at 80–90% of confluency, seeded (2 × 10⁶ per well) in 6-well plates and incubated at 37 °C in a 5% CO₂ atmosphere until cell attachment. Cells were serum-starved 12–16 h prior the stimulation, using serum-free DMEM containing 0.25% BSA and incubated at 37 °C in a CO₂ incubator. After 24 h of treatment, medium was collected and centrifugated at 10,000× g to remove debris and dead cells and analysed for the concentration of IL-8, G-CSF, TNF-α and MIP-1β, by using the Bioplex Multiplex human cytokine assay (Bio-Rad), as indicated by manufacturer’s instructions. As a multiplexed assay, the Bioplex assay can simultaneously detect more analytes in a single sample [82 (link)].