Molecules and Cells

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Fig. 2.

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Fig. 2. (A) The anesthetized mouse is cannulated via the portal vein and the line is connected. (B) The pump is started, the infrahepatic inferior vena cava (IVC) is cut off, and the chest of the mouse is opened. (C) The supraheptic IVC is cannulated via the right atrium and the infrahepatic IVC is clamped. The liver is perfused with PBS to eliminate blood. (D) The line is connected to the suprahepatic IVC catheter, followed by closed circulation for up to 2 h. The resulting liver samples can be ground to isolate hepatic immune cells, or further digested with collagenase to isolate each hepatic cell type (e.g. hepatocytes, hepatic stellate cells (HSCs), Kupffer cells (KCs), and liver sinusoidal endothelial cells (LSECs)). (E) Perfusion of the liver with collagenase solution, followed by in vitro digestion and filtering of the cell suspension through a 70-μM mesh. (F) Pelleting of hepatocytes by low-speed centrifugation (50×g for 5 min at RT, twice). Hepatocytes can be further purified by 40% Percoll-gradient centrifugation (1,000×g for 10 min at 40°C). Supernatants containing non-parenchymal cells are pelleted by high-speed centrifugation (590×g for 10 min at 4°C, twice) and further separated by density gradient centrifugation (1,600×g for 17 min at 4°C). HSCs can be further purified by retinoid-based FACS sorting. KCs and LSECs can be further purified using F4/80+ and CD146+ MicroBeads, respectively, followed by MACS.
Mol. Cells 2019;42:45~55
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