Isolation and Stimulation of Kupffer Cells

Primary hepatic stellate cells, Kupffer cells and hepatocytes were isolated as previously described and expression of Clec4n was measured by real-time qPCR.25 (link) Kupffer cells were isolated from C57BL/6 and Clec4n^–/– mice and cultured with RPMI 1640 (Thermo Fisher Scientific) containing 10% FBS for 4 hours. After 12 hours of starvation in RPMI1640 (without FBS), Kupffer cells were stimulated with M. restricta lysate (40 µg/mL) or PBS (control) for 8 hours. M. restricta lysate was prepared by washing centrifugation pellets of M. restricta with PBS twice, dissolving in 1 mL PBS and then disrupting using 0.5 mm glass beads in a MINI-BEADBEATER BIOSPEC PRODUCTS twice for 30 seconds with 3 minutes cooling on ice between runs. The concentration of M. restricta lysate was 0.15 mg/mL, measured by bicinchoninic acid assay.