Isolation and Analysis of Intrahepatic Lymphocytes

Intrahepatic lymphocytes (IHLs) were isolated as previously described (30 (link)). In brief, liver was perfused with 10 ml PBS, minced and digested with RPMI 1640 containing 0.05% collagenase IV (Roche, Indianapolis, IN) at 37 °C for 30 min. After digestion, cell suspensions were passed through 70 μm cell strainers, followed by a centrifugation over a 30/70% discontinuous Percoll density gradient (Sigma, St. Louis, MO) at 400 g at room temperature for 30 min. The cells were collected from the interphase, washed, resuspended in complete RPMI 1640, and the total number of IHLs per liver was counted. The relative percentages of immune cell populations were then analyzed by flow cytometry, and the absolute numbers of these lymphocyte subpopulations per liver were calculated according to their percentages and total IHL numbers in each liver.