Sector imager 6000 plate reader

Plasma concentrations of TNF, IFN-γ, IL-2, IL-4, and IL-8 were measured using a V-PLEX Custom Human Proinflammatory Panel 1 kit (K151A9H), plasma concentrations of IL-17 using a Human IL-17A S-PLEX kit (K151C3), and plasma concentrations of TNFR1 and TNFR2 using Human TNFR-I ultrasensitive (K151BIC) and Human TNFR-II ultrasensitive (K151BJC) kits (All from Mesoscale Discovery (Meso Scale Diagnostics, Rockville, MD, USA)). Samples were diluted in Diluent-41 and analyzed in duplicate according to the manufacturer’s instructions. Analysis was performed on a SECTOR Imager 6000 plate reader and MSD Discovery Workbench software 4.0 (Mesoscale Discovery, Meso Scale Diagnostics, Rockville, MD, USA) as previously described [48 (link),49 (link)]. Sample replicates with coefficient of variation (CV) values > 25% in individual analyses were excluded. Lower levels of detection (LLOD) were: IL-2 = 0.045–0.058 pg/mL; IL-4 = 0.016–0.025 pg/mL; IL-8 = 0.023–0.045 pg/mL; IL-17 = 49.0–87.8 pg/mL; IFN-γ = 0.593–0.951 pg/mL; TNF = 0.057–0.153 pg/mL; TNFR1 = 0.264–1.900 pg/mL; TNFR2 = 0.698–2.140 pg/mL. For protein levels below LLOD, a value of 0.5 LLOD was used for statistical analysis. Cytokine measurements were performed by a technician blinded to the clinical data.

Plasma concentrations of TNF were measured using V-PLEX Human Proinflammatory Panel 1 and plasma concentrations of TNFR1 and TNFR2 were measured using Human TNFR-I ultra-sensitive kit and Human TNFR-II ultra-sensitive kit. Interleukin (IL)-6 and vascular endothelial growth factor (VEGF-A) were measured using the V-Plex Human IL-6 kit and V-Plex Human VEGF kit (all from Mesoscale Discovery, Rockville, MD, USA). Serum concentrations of CCL2 were measured using a V-PLEX human MCP-1 kit (Mesoscale Discovery). Samples were diluted in Diluent-41 and analyzed in duplicate according to the manufacturer’s instructions. Analysis was performed on a SECTOR Imager 6000 plate reader and MSD Discovery Workbench software was used for analysis (Mesoscale Discovery). Sample replicates with coefficient of variation (CV) values >25% in individual analyses were excluded. None of the measurements were below the lower level of detection.