Purification of Mouse Liver Cells

Mouse liver cells purification protocol was adapted from refs. ^{31 (link),32 (link)}. Briefly, liver cells were isolated following a two-step protocol of Pronase/Collagenase digestion. Firstly, the liver was perfused with HBSS (Sigma #H6648) containing 0.2 mg/mL EDTA. Secondly, a perfusion with 0.4 mg/mL Pronase (Sigma #P5147) and 2 mg/mL Collagenase Type II (Worthington #LS004196) in HBSS was performed. Finally, the liver was perfused with HBSS containing 0.4 mg/mL Pronase, 2 mg/mL Collagenase Type II and 0.1 mg/mL DNase I (Roche #R104159001). The liver was minced and further digested with HBSS containing 0.4 mg/mL Pronase, 2 mg/mL Collagenase Type II and 0.1 mg/mL Dnase I for 25 min with shaking at 37 °C. To stop digestion, DMEM (Thermo Fisher #31966047) was added. The resulting liver cell suspension was filtered and washed three times through centrifugation at 300 g for 4 min in 2% FBS-PBS. The suspension was then subjected to density gradient centrifugation using 20% Percoll (GE Healthcare #17-0891-01) to remove dead cells. Resuspension of the cells in ACK lysis buffer (Thermo Fisher #A1049201) allowed the lysis of red blood cells. Cell suspension was then centrifuged and resuspended in 2% FBS-PBS to proceed with scRNA-seq analysis using 10X Genomics Chromium Single-Cell 3’ according to the manufacturer’s instructions.