After rinsing the slides with PBS containing 0.05% Tween 20, HRP-conjugated goat anti-rat IgG (Life Technologies) for the rat primary antibodies was used to visualize the antibody-antigen reaction. The immunostained slides were observed and photographed with a Leica DM5000B phase contrast microscope equipped with a digital camera system. Fluorescence-labeled polystyrene microbeads (1.0 µm diameter, #17154, Polysciences, Inc., Warrington, PA) were

diluted to 1:800 in the growth medium and added to the primary Kupffer cells, KUP5, MG6 and BMDM cells seeded in 60 mm non-tissue culture grade plastic dishes (5×105 cells/plate). After 1 and 2 h, the cells in the plastic dishes were rinsed with PBS three times to remove nonphagocytosed beads [22] and [23], harvested with TrypLE Express and fixed with 3.7% formalin in PBS at room temperature for mTOR inhibitor 15 min. After washing with PBS, cells were suspended in 0.5 ml of Iso Flow (Beckman Coulter, Fullerton, CA) and analyzed with check details a flow cytometer (Epics XL-MCL, Beckman Coulter) for phagocytosis of

the fluorescence-labeled microbeads. The primary Kupffer cells, KUP5, MG6 and BMDM cells were seeded in a 12-well cell culture plate at a density of 2×105 cells/well. The next day, the medium was replaced by growth medium containing lipopolysaccharide (L3129, Sigma-Aldrich) at 0.1–1.0 µg/ml. After incubation for 24 h at 37 °C, the culture supernatant was collected, filtered with a membrane filter (0.20 µm pore size, Millipore Millex) and stored at −80 °C until use. Aliquots of the samples were assayed using mouse cytokine ELISA kits (R&D Systems, Minneapolis, MN), according to the manufacture’s instructions. The experiments were independently performed three times and the cytokine concentrations in the culture supernatant are expressed as the mean value±SEM. The KUP5 and MG6 cells were seeded in eight-well chamber PAK6 glass slides at the density of 2×104 cells/well along with the growth medium containing recombinant

mouse GM-CSF (415-ML, R&D Systems, Minneapolis, MN) at 10 ng/ml. After incubation for 3 days, the cells were washed with PBS, fixed with 95% ethanol and 1% acetic acid. After rinsing with PBS, the slides were mounted with mounting medium containing DAPI and photographed with a Leica DM5000B fluorescent microscope system equipped with a digital camera. We established an immortalized Kupffer cell line (KUP5) from a mixed primary culture of adult C57BL/6 mouse hepatocytes by a retrovial transduction of the c-myc oncogene. These macrophage-like cells probably have originated from Kupffer cells, which were contaminants in the parenchymal hepatocyte fraction at the start of the culture. These cells proliferated vigorously in response to the specific culture environment provided by the morphologically transformed mouse hepatocytes [13], and thus were susceptible for the infection by the retroviral vector.