Msd u plex platform

The supernatant of the MLR co‐cultures was used for quantification of IFN‐γ, TNF‐α, IL‐1β, IL‐6, IL‐8, IL‐10, IL‐12 (p70), IL‐13, IL‐17A, IL‐33 using a MSD U‐plex platform (Mesoscale Discovery, Rockville, USA), according to the manufacturer's instructions.

PBMC culture supernatants were analyzed for cytokine concentrations using the MSD U-plex Platform, U-plex Biomarker group 1 (human) Multiplex Assay using a U-plex 5-assay 96 well sector plate (MESO Scale Discovery, Rockville MD USA) with the MSD sector imager 2400 (MESO Scale Discovery) or the Mesoscale SQ 120. PBMC cultures stimulated with anti-CD3 plus anti-CD28 antibodies, or with the corresponding isotype control were diluted 1:10 and 1:2, respectively, for determination of interferon-γ and interleukin (IL)-10, IL-13 and IL-17A concentrations. PBMC cultures stimulated with LPS, or its negative control, were diluted 1:40 and 1:2, respectively, for determination of tumor necrosis-factor (TNF)-α, IL-1β, IL-6 and IL-10 concentrations. Mean concentrations (μg/L) of duplicate wells were used for analysis.

Cytokine analyses were performed as previously described^{33 (link)}. Single cell suspensions of leukocytes isolated from the liver (10⁵ per well) or sorted NKT cells from the liver (2.5 × 10⁵ per well) were cultured in complete RPMI 1640 medium supplemented with 10% FCS, 100 U/ml penicillin/streptomycin, 2.5% sodium bicarbonate (7.5% solution), 1 mM sodium pyruvate and 69 µM 1-thioglycerol unstimulated or activated with an anti-CD3 antibody (4 µg/ml, clone 154-2C11, BD Biosciences). The supernatants were collected after 24 h in culture and stored at −80 °C before being analyzed for cytokines using the mouse Th1/Th2/Th9/Th17/Th22/Treg Cytokine 17-plex Mouse ProcartaPlex Panel (Invitrogen) according to the manufacturer’s instructions.
Plasma was collected using EDTA-coated microcentrifuge tubes and centrifuged for 15 min at 1000 × g. The cleared plasma was collected and stored at −80 °C before being analyzed for cytokines. For cytokine measurements in plasma or culture supernatants of sorted NKT cells, the MSD U-PLEX platform (Meso Scale Diagnostics, Rockville, MD, USA) was used according to the manufacturer’s instructions.