Isolation and Immunophenotyping of Liver Cells

Liver tissue was collected and incubated in DMEM (Thermo Fisher Scientific; catalogue number 11965118), 10% FBS (GE Healthcare Life Sciences, Melbourne, VIC, Australia; catalogue number SH30084.03) and 1 mg/mL collagenase (Sigma-Aldrich; catalogue number C5138) for 1 h [32 (link)]. Afterward, leucocyte suspensions were obtained, and immunostaining was performed as described [22 (link),33 (link)]. Cells were stained with LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit (Thermo Fisher Scientific; catalogue number L34962, 1/200 dilution). Antibodies are listed in Table 2. Cells were analysed on a custom 10-laser LSR II (BD Biosciences, Franklin Lakes, NJ, USA). Flow cytometry data were analysed with FlowJo software version 9.9 (TreeStar, Ashland, OR, USA) [33 (link)].