Isolation and Purification of Viable Hepatocytes

Viable hepatocytes were collected using a method adapted from Ben-Moshe et al. (2019) (link). In details, isolated hepatocytes were resuspended in 25mL EBSS then mixed with 22.5ml Percoll (Sigma) and 2.5mL 10xPBS to remove dead cells. The mixture was centrifuged at 600rpm for 10 minutes, supernatant was discarded, and the cells were resuspended in cold FACS buffer (2mM EDTA, 10% FBS in 1x PBS). 100μl of 10⁶ cells were aliquot into FACS tubes and .25μg FcX blocking solution (Biolegend) was added. Cells were incubated on ice for 30 minutes. Next, 2ml of FACS buffer was added to each tube, and centrifuged at 1,000rpm for 3 minutes to wash the cells. Cells were resuspended in 100μl FACS buffer and stained with APC-CD31(BioLegend, cat: 102509), PE/Cy5-CD45 (BioLegend, cat: 103109), APC/Cy7-CXCR2 (BioLegend, cat: 149313) in a dilution of 1:100.