Proinflammatory panel 1 human kit

Serum interleukin 1 receptor antagonist (IL-1ra) concentrations were measured with Human IL-1ra/IL-1F3 Quantikine ELISA kits (R & D Systems, Minneapolis, USA). Serum TNF and IL-6 concentrations were measured with Proinflammatory Panel 1 Human Kit (Meso Scale Diagnostics, Maryland, USA), and C-reactive protein (CRP) was analyzed with Vascular Injury Panel 1 Human Kit (Meso Scale Diagnostics, Maryland, USA). Human Leptin, Insulin Kit (Meso Scale Diagnostics, Maryland, USA) was used to analyze serum leptin concentrations. All serum samples were tested in duplicate. The intra- and inter-assay coefficients of variation (CV) were 3.1% and 15.6% (IL-1ra), 4.2% and 7.0% (IL-6), 3.1% and 12.1% (TNF-a), 2.5% and 10.0% (CRP), and 6.6% and 8.9% (leptin), respectively.

Cytokine levels were quantified in apical and basolateral media using an electro-chemi-luminescence-based Meso Scale Discovery (MSD) platform exploiting the V-PLEX Proinflammatory Panel 1 Human Kit. This kit provides assay-specific components for the quantitative determination of IFNγ, IL1β, IL2, IL4, IL6, IL8, IL10, IL12p70, IL13, and TNFα. Preparation of samples and detection plates were performed following the manufacturer’s instructions (Meso Scale Diagnostics, Rockville, MD, USA) with few adaptations. In short, a series of 9 concentrations of standards in duplo, together with the samples were added to the plate. Plates were incubated while shaking at room temperature for 2 h. After incubation, plates were washed twice, and the respective detection antibody mixture was added to each well. Again, plates were incubated while shaking at room temperature for 2 h. Afterwards, plates were washed and 2X Read buffer was added to each well. Plates were read on the MSD Plate reader (MESO QuickPlex SQ 120, Meso Scale Diagnostics, Rockville, MD, USA). Protein concentrations were determined using the MSD Discovery Workbench 4.0 analysis software. Results are presented as fold change compared to the control for each culture.

From each blood sample for each time point 250 µl of the cell suspension was centrifuged at 4 °C for 5 min with 1,000 G. The supernatant was collected and frozen for cytokine and chemokine determination at − 80 °C. Levels of IL-1β, IL-2, IL-4, IL-6, IL-8, IL10, IL-12p70, IL-13, TNF-α, and IFN-γ (V-PLEX Proinflammatory Panel 1 Human Kit), Eotaxin, Eotaxin-3, IL-8, IL-8 (HA), IP-10, MCP-1, MCP-4, MDC, MIP-1α, MIP-1β, and TARC (V-PLEX Chemokine Panel 1 Human Kit) and GM-CSF, IL-1α, IL-5, IL-7, IL-12/IL-23p40, IL-15, IL-16, IL-17A, TNF-β, and VEGF-A (V-PLEX Cytokine Panel 1 Human Kit) were analysed via an electrochemiluminescent immunoassay according to the manufacturer’s instructions (Meso Scale Discovery (MSD), Gaithersburg, MD, USA) using the Meso Quick Plex SQ 120. Raw data were analysed using the Discovery Workbench 4.0 software (MSD).