V plex proinflammatory panel 1 human kit

Blood samples for the quantification of proinflammatory cytokines were kept on ice and rapidly sent to the research laboratory to be centrifuged at 1100× g (10 min at 4 °C) within 1 hour of sampling. Plasma was then aliquoted and stored at −80 °C until use. Levels of inflammatory markers INF-γ, IL-1β, IL-6, and TNF-α were quantified by electrochemiluminescence immunoassays using the V-PLEX Proinflammatory Panel 1 Human Kit, QuickPlex SQ120 Imager, and WORKBENCH software (MSD^®, Rockville, MD).

Multiplex assays to quantitatively measure levels of ten different cytokines (IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13 and TNF-α) in the plasma were performed using the MSD V-PLEX Proinflammatory Panel 1 (human) kit according to the manufacturer’s instructions (lot no. K0081665). Briefly, 96-well plates were washed before adding serum diluted 1:4 in diluent and calibrators. After incubation, plates were washed and detection antibodies were added. Plates were washed and read immediately using the MESO QuickPlex SQ 120 system. Data were generated by the Methodological Mind software and analyzed with the MSD Discovery Workbench v.4.0 software. The presented data were adjusted for any sample dilution.

Cytokine levels from unstimulated and stimulated blood samples were quantified using the V-PLEX Proinflammatory Panel 1 (human) Kit (MSD, K15049D). Cytokine quantification was done according to the manufacturer’s guidelines with one modification, where 100 μl sample was added directly onto the plate without dilution. C-reactive protein levels were quantified using the kit following the manufacturers guidelines (MSD, K151STD) using a 1000-fold dilution. Sensitivity of each assay is shown in Supplementary Table 1. Specificity of each assay was <0.6%.

Whole blood was collected into 4-mL lithium-heparin containing tubes (Greiner Bio-One, 454029). For the whole blood stimulation, whole blood was diluted 1:10 with Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham (Sigma-Aldrich) supplemented with 10% foetal calf serum and 5% penicillin streptomycin. Of the diluted mixture, 500 μL was aliquoted to a 24-well plate. Each well was stimulated with 5 μL of the stimulant Escherichiacoli K12 lipopolysaccharide (LPS) which is a Toll-like receptor 4 (TLR4) agonist. Dysregulation of signalling pathways associated with TLR4 receptors is one of the most replicated findings in relation to alcohol and inflammatory processes.⁴⁰ (link) The blood was harvested after 24 h of incubation at 37 °C and stored at −80 °C for later analysis. The following cytokines were analysed both in unstimulated and TLR4 agonist-stimulated conditions: Tumour Necrosis Factor α (TNF-α), Interferon-γ (IFN-γ) and Interleukins (IL-1β, IL-2, IL-10, IL-6, IL-8). Cytokine levels were quantified using the V-PLEX Proinflammatory Panel 1 (human) Kit (MSD, K15049D). Cytokine quantification was done according to the manufacturer's guidelines with one modification, where a total of 100 μL (50 μL in duplicate) sample was added directly onto the plate without dilution (applied for both stimulated and unstimulated conditions).