Il 6 elisa kit

Mouse livers were perfused with 30 mL of HBSS with 0.5 mM of EDTA and 25 mM of HEPES and then dissected and digested with Collagenase IV (Roche). Single cells were pelleted and resuspended in 450 μl of PBS containing 2% FBS after red blood cells were removed. Kupffer cells were purified using anti-F4/80 microbeads (Thermo Fisher Scientific, #8802-6863-74) with an LS column (Miltenyi Biotec,130-042-401). The enriched liver macrophages (consisting of 80-90% F4/80^hiCD11b^intGr1^low KCs and 10–20% of F4/80^intCD11b^hiGr1^low MDMs) were seeded into 6-well plates pre-coated with collagen. After plating for 1-2 hrs, cells were washed with PBS and cultured in a fresh medium overnight for glycolysis analysis, ROS, and cytokine production. Fresh KCs from DPI-treated or vehicle-treated mice were stimulated with or without 100 ng/mL LPS for 6 h and cytokines TNF-α, IL-1β, and IL6 in the supernatant were measured by the TNF-α ELISA kits (Dakewe, # 1217202), the IL-1β ELISA kit (Dakewe, #1210123) and IL-6 ELISA kit (Dakewe, #1210602), respectively, according to the manufacture’s manuals. Fresh KCs from B6 mice were first treated with or without DPI at indicated concentrations for 24 h and then stimulated with 100 ng/mL LPS in fresh medium (without DPI) for ROS and cytokine analysis.

The quantification of cytokines in mice serum was performed using an enzyme-linked immunosorbent assay (ELISA): GM-CSF kit (#1217302, DAKEWE), VGEF ELISA kit (#1217342, DAKEWE), TGF-β ELISA kit (#1217102, DAKEWE) and IL-6 ELISA kit (#1210602, DAKEWE), following the manufacturer’s instructions. The absorbance was analysed using an i3 Paradigm multi-label reader (Molecular Devices).

Serum levels of inflammatory cytokines were measured using the mouse TNFα ELISA kit (Dakewe Biotech, 1217202) and IL-6 ELISA kit (Dakewe Biotech, 1210602) according to the manufacturer's protocols.