Nucleofection in Rat Kupffer Cells. Freshly isolated
Kupffer cells were transfected using the Amaxa mouse macrophage Nucleofector kit according to the instructions of the manufacturer using the Y-001 program (Lonza, Cologne, Germany), except for the following modifications. Samples were processed individually, and the entire nucleofection procedure for each sample was
completed in less than 5 minutes. For each nucleofection sample, 2 x 10^6 Kupffer cells were centrifuged for 10 minutes at 300g. The pellet was washed with 1 mL phosphate-
buffered saline (PBS), collected at 300g for 5 minutes, and then resuspended in 105 uL nucleofector solution and transferred to 1.5-mL Eppendorf tubes for a final concentration of approximately 2 x 10^6 cells/100 uL. Cells were then treated or not with 2.0 ug specific or
scrambled siRNA (siRNA sequences are provided in Supplemental Materials), transferred into the electroporation cuvette, and placed in the Nucleofector device. After nucleofection, cells were immediately removed from the cuvette and plated in a 96-well plate (150 uL/well) at
0.5 x 10^6 cells/well. After 4 hours, the cell culture medium was replaced with fresh medium with or without 1 ug/mL gAcrp or 10 ng/mL IL-10 for 18 hours and then treated with or without LPS or IL-10, as described in the
figure legends.