TITLE: Mouse 2- Step Collagenase Liver Perfusion protocol 
AUTHORS: Michael Cheng, Xue-Zhong Ma, Chao Jiang, Ian McGilvray, Sonya Macparland

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Analyzing the mouse liver requires optimal cell recovery from the tissue in terms of quality (viability) and quantity. Harsh dissociation methods can damage fragile cell populations, resulting in low viability and difficulty in cell culture. We have developed a gentle surgical and enzymatic technique based on the two-step collagenase perfusion protocol for single-cell analysis and primary cell culture.</div></div>

[STEPS]
?. [Preparation]
Anesthetize mouse with inhalational anesthesia (4-5% isofluorane) in the induction chamber
?. [Laparotomy]
Midline incision: cut open the abdomen from the pubic symphysis to the xiphoid process. Flip over the intestines to locate the liver and its portal vein.Please refer to Figure 14.4.1 of the following publication:
?. [Cannulation]
Cannulate the portal vein with the 22-gauge catheter. Secure it cannulation by suture or a micro serrefine.
?. [Perfusion]
Perfuse liver with calcium-free buffer without collagenase:   of Hanks' Balanced Salt Solution (HBSS) without calcium or magnesium +  at a flow rate of 2 mL/min until fluid drained from the IVC is no longer red (containing blood). This is done with a pump with no recirculation.
10 mL
[EGTA]
This flow rate (2 mL/min) is slower than other liver perfusion protocols. We found that a slower perfusion rate results in hepatocytes with higher quality and quantity and therefore suitable for analyses such as single-cell RNA sequencing.
?. [Harvest]
Excise the dissociated liver out of the animal and place it in DMEM containing 10% FBS, which inactivates the collagenase enzyme.
?. [Cell Isolation]
Gently cut the excised liver with a scalpel to release the dissociated cells.
Good dissociation can be visualized by abundance of cells released (in pink) with little cutting. Shake the tissue gently to release cells. Avoid tearing the tissue with the scalpel (horizontal movement); only cut the tissue vertically.
?. [Cell Enrichment]
Enrich dissociated cells for hepatocytes by centrifugation   for 5 minutes. The supernatant can be further purified in the next step. Resuspend the pellet (hepatocyte-rich) with DMEM.
0 Room temperature
?. [Cell Enrichment]
Enrich supernatant for non-parenchymal cells (NPC) by centrifugation  for 5 minutes. Discard the supernatant. Resuspend the pellet (NPC-rich) with DMEM.
0 Room temperature
?. [Preparation]
Prepare the abdomen for surgery: shaved with a trimmer, prepared with iodine-based solution and alcohol-based solution, and draped in a sterile fashion.
?. [Preparation]
Heparinize the animal intraperitoneally by injecting  of heparin:
Preventing blood clotting improves cell recovery.
?. [Preparation]
Once the animal is anesthetized, transfer it onto a homeothermic warming pad. The animal will receive 1-3% isofluorane via face-mask inhalation fo the remaining steps.
?. [Perfusion]
Perfuse liver with calcium-containing buffer with collagenase:  of Hanks' Balanced Salt Solution (HBSS) with calcium and magnesium +  type IV collagenase at the same flow rate (2 mL/min). This is done with the same pump with no recirculation.
10 mL
?. [Cannulation]
Collect blood: collect blood from the inferior vena cava (IVC) with a 20-gauge needle.
?. [Cannulation]
Cut the IVC and proceed to the next step immediately. Cutting the IVC allows blood and perfusate to drain.
?. [Cell Isolation]
Filter dissociated cells with a 70 µm filter: