We use collagenase to digest hepatocytes in the process of liver perfusion. The activity of collagenase is based on the lot number, and not all lots are good enough to use them for liver perfusion,it should be established first.

When isolating mouse hepatocytes, digesting the liver tissue effectively is crucial to obtain high-quality cells. Here's a step-by-step guide to help you digest the liver well:

Preparation

1. *Choose the right enzyme*: Collagenase is the most commonly used enzyme for liver digestion. You can use either collagenase type I or type IV.

2. *Prepare the digestion buffer*: Typically, a buffer containing collagenase (0.5-1 mg/mL), calcium chloride (5-10 mM), and HEPES (10-20 mM) is used.

Digestion Steps

1. *Perfuse the liver*: Perfuse the liver with a calcium-free buffer to remove blood and debris.

2. *Digest the liver*: Infuse the digestion buffer into the liver through the hepatic portal vein. You can use a perfusion pump to control the flow rate.

3. **Inc

Preparation

1. *Choose the right enzyme*: Collagenase is the most commonly used enzyme for liver digestion. You can use either collagenase type I or type IV.

Digestion Steps

1. *Perfuse the liver*: Perfuse the liver with a calcium-free buffer to remove blood and debris.

2. *Digest the liver*: Infuse the digestion buffer into the liver through the hepatic portal vein. You can use a perfusion pump to control the flow rate.

3. *Incubate the liver*: Place the liver in a water bath at 37°C for 10-15 minutes to allow the collagenase to digest the connective tissue.

4. *Gently shake the liver*: Gently shake the liver every 2-3 minutes to help distribute the enzyme and facilitate digestion.

Tips for Effective Digestion

1. *Use the right concentration of collagenase*: Too little collagenase may result in incomplete digestion, while too much can cause cell damage.

2. *Monitor the digestion*: Check the liver's texture and appearance during digestion. It should become soft and fragile.

3. *Avoid over-digestion*: Stop the digestion process when the liver is fully digested. Over-digestion can lead to cell damage and death.

4. *Use a gentle shaking motion*: Avoid vigorous shaking, which can damage the cells.