Monoclonal antibodies against cell surface markers

Single-cell suspensions from the hepatic MNCs were incubated with monoclonal antibodies against cell surface markers (BD Biosciences Pharmingen, San Diego, CA). PE-labeled CD1d-α-galactosylceramide tetramer [3] (link) and FITC-, PE-, or APC-conjugated Abs specific for mouse CD3 (145-2C11), CD49b (DX5), Mac-1 (M1/70), and Gr-1 (RB6-8C5) were used for flow cytometric analyses. For the detection of intracellular IFN-γ in hepatic MNCs, the cells were prepared from wild-type and LECT2-deficient mice at 3 h after LPS/d-GalN injection. Hepatic MNCs were cultured in RPMI-1640 supplemented with 10% FBS and 10 µg/ml of brefeldin A (Sigma) for 2 h. The cells were then stained with FITC-conjugated anti-CD3 and PE-conjugated anti-DX5 antibodies, fixed, permeabilized, and again stained with APC-conjugated anti-IFN-γ (XMG1.2) antibody by using a BD cytofix/cytoperm kit (BD Biosciences Pharmingen) according to the manufacturer's instructions.