2.5. Isolation and culture of Kupffer cells

Kupffer cells were obtained from livers of PTP1B^+/+ and PTP1B^−/− mice. For Kupffer cells isolation, the supernatant from the first centrifugation of the hepatocyte isolation protocol was collected and centrifuged twice at 50×g for 5 min to discard the pellet with the remaining hepatocytes. The latest supernatant was centrifuged at 300×g for 5 min at 4 °C and the pellet containing the Kupffer cells was resuspended in attachment media. Kupffer cells were separated by two-step Percoll gradient method. After centrifugation at 800×g for 10 min (with breaks off), Kupffer cells were enriched between 25% and 50% Percoll. Finally, Kupffer cells pellet was washed with PBS, centrifuged twice at 500×g for 10 min at 4 °C to wash out the residual percoll solution, and cells were resuspended in DMEM supplemented with 10% heat inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM glutamine.