Gal 9

NK-92MI cells were cultured at 37 °C with 5% CO₂ in alpha minimum essential medium (αMEM, Gibco, Waltham, MA, USA) supplemented with 12.5% horse serum and 12.5% fetal calf serum (FCS) (Gibco), containing 0.2 mM inositol (Sigma, Le Chesne, France), 0.1 mM 2-β mercaptoethanol (Gibco), 0.02 mM folic acid (Sigma), 100 U/mL penicillin, and 100 µg/mL streptomycin. To evaluate the effect of different serum additives, we added 10% FCS or 10% human AB serum (ABS) (Gibco) to the serum-free culturing medium and incubated the cell for 24 h at 37 °C. Treatment with human E. coli-derived recombinant Galectin-9 (Gal-9, Cat No:20-45GA, R&D Systems, Minneapolis, MN, USA) was performed in 30 and 100 nM concentrations for 24 h at 37 °C. K562 cells were maintained in Roswell Park Memorial Institute 1640 medium (RPMI, Lonza, Verviers, Belgium) supplemented with 10% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin (Lonza).

Bone marrow-derived MSCs from C57/BL6 mice were purchased from Cyagen Biosciences (Sunnyvale, Calif). The culture process was conducted according to the manufacturer’s instructions. Sixth- to eighth-generation MSCs were collected for use. At 3 h after CLP, the mice were injected with 10⁶ MSCs, IgG (BD Biosciences, San Diego, CA), Gal-9 (100 µg/mouse, R&D Systems, Minneapolis, MN), or soluble Tim-3 (100 µg/mouse, eBioscience, San Diego, CA) via the tail vein.

Standard ELISA methods were used to measure concentrations of Gal-9 (R&D Systems), cytokines and chemokines such as IL-12p40, IL-12p70, MIP-1β (macrophage inflammatory protein [MIP]), monocyte chemoattractant protein 2 (MCP-2), tumour necrosis factor-alpha (TNF-α), IL-6, transforming growth factor-beta1 (TGF-β1), IL-18, IL-23, IL-27, and IL-1β (R&D Systems) and IFN-α (Verikine™ Human IFNα ELISA Kit, PBL Assay Science, Piscataway, NJ, USA).