Hello,

I have tried to isolate hepatocyte from mouse 5 times. I used two-step isolation methods, perfusion and isolation, and performed 60% percoll gradient centrifugation. However, I did not get any pellet of cells (less than 0.7 million cells only) and the viability was very poor. I suspect several things that caused this problem.

1. collagenase type: I used collagenase D (0.2 mg/ml)

2. pH of buffer: it was pH 7.42

3. perfusion rate (flow rate): it was 800 ml/hr

4. percoll percentage: 60%

If there is any problem among the list, please point out for me.

Or, if there is something that I would have missed or if there is any secret steps, please tell me.

I really need your help. Please! Thank you!