Discovery workbench software v 4

Interleukin 1 alpha and IL-18 cytokine level of each well was calculated according to the standard curves by DISCOVERY WORKBENCH Software v 4.0 (Meso-Scale Discovery, Rockville, MD, United States). The relative increase or decrease of cytokine levels was normalized to the fold-change over 1% DMSO (vehicle control). The means and SDs were calculated by three replicate plates and reported as fold-change over vehicle control.
Statistical analyses were performed using GraphPad Prism software 7.0 (San Diego, CA, United States). Student t-test with the Holm–Sidak posttest was used for statistically significant analyses that involved three experimental groups.

Liver tissue was homogenized by sonication in 20 mM TRIS (pH 7.5), 2 mM EDTA, 10 mM EGTA, 1% Triton X-100, and 250 mM sucrose with HALT™ Protease and Phosphatase Inhibitor (Thermo Fisher Scientific), then centrifuged for 10 min at 10,000× g. Supernatants were collected, protein concentration was measured by BCA method (Thermo Fisher Scientific). CXCL2 was detected using the MesoScale Discovery Mouse MIP-2 V-Plex Kit as per manufacturer’s instructions (MesoScale Discovery, Rockville, MD), read on the MESO Sector S 600 Instrument, and analyzed with Discovery Workbench Software, v 4.0 (Meso Scale Discovery). PAI-1 was detected using the Mouse PAI-1 ELISA Kit according to the manufacturer’s instructions (Catalog No. EMSERPINE1, Thermo Fisher Scientific).

PBMC CDC assays were performed as follows. Human PBMCs from eight healthy donors (isolated from buffy coats through centrifugation using Leucosep tubes according to the manufacturer’s instructions) were incubated with antibodies in culture medium (Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 1 U ml⁻¹ penicillin and 1 µg ml⁻¹ streptomycin, Lonza) and 20% (final concentration) pooled NHS (Sanquin) as a source of complement, in three individual samples per antibody. After 20–24 h incubation at 37 °C/5% CO₂, culture supernatant was collected and stored at −20 °C until further analysis. Cytokine quantitation was performed on the culture supernatants using the U-PLEX Proinflam Combo 1 Human kit and Discovery Workbench software v.4.0 (Meso Scale Diagnostics), according to the manufacturer’s instructions. Two donors were excluded based on a high induction of cytokine production by isotype controls.
The gating strategy used to define cell populations was essentially the same as described for whole blood cytotoxicity analysis, and is detailed further in Supplementary Fig. 7. In addition, PBMCs were stained for cell surface markers CD25 (CD25-PE, eBioscience) and CD69 (CD69-FITC, BD Biosciences). Staining was performed for 30 min at 4 °C and analyzed on an LSR Fortessa X20 flow cytometer (BD Biosciences).