Multiplex panel

The plasma cytokine levels of ACLF patients were measured using a multiplex panel (Bio-Rad, Hercules, CA), according to the manufacturer’s instructions. PGE2 was measured using the Prostaglandin E Metabolites ELISA kit (Cayman Chemical, Michigan, USA). Both measurements were performed according to the manufacturer’s instructions. The detection limits were as follows:IL-1β, 17.3 pg/ml; IL-1rα, 15 pg/ml; IL-2, 16.5 pg/ml; IL-4, 11 pg/ml; IL-5, 27 pg/ml; IL-6, 24.8 pg/ml; IL-7, 11 pg/ml; IL-8, 14.5 pg/ml; IL-9: 21 pg/ml; IL-10: 33.3 pg/ml; IL-12: 15.5 pg/ml; IL-13: 13.5 pg/ml; IL-5; 57.3 pg/ml; IL-17: 24.8 pg/ml; eotaxin, 17 pg/ml; FGF basic, 14 pg/ml; G-CSF, 36.3 pg/ml; GM-CSF, 16 pg/ml; IFN-γ, 15.5 pg/ml; IP-10, 14.5 pg/ml; MCP-1, 18.5 pg/ml; MIP-1α, 8.8 pg/ml; PDGF-bb, 32.5 pg/ml; MIP-1β, 31.5 pg/ml; RANTES, 15.8 pg/ml; TNF-α, 12.5 pg/ml; VEGF, 109.3 pg/ml; PGE2, 2 pg/ml.

In vitro, PBMCs (2 × 10⁵/well) were stimulated with LPS (100 ng/ml; Sigma-Aldrich, St. Louis, MO) for 72 h in the presence or absence of AH6809 (150 μM) or butaprost (50 μM; Sigma) or DMSO in 10% FBS RPMI medium. Next, the supernatants were collected and stored at − 80 °C immediately. Cytokines in the supernatants were measured using a multiplex panel (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. The limitations of detection have been mentioned above.

Whole blood samples from participants were collected within two days after study enrollment. After centrifugation, plasma was obtained and stored immediately at − 80 °C. Plasma samples (25μL) were used to measure suPAR using an enzyme-linked immunosorbent assay (ViroGates, Denmark), and 20μL of plasma sample was used to measure cytokines using a multiplex panel (Bio-Rad, Hercules, CA), according to the manufacturer’s instructions. The detection limits were in the supplementary methods (Additional file 1).