Table 1 Frequent causes unsuccessful hepatocyte isolation.
From: Improved isolation of murine hepatocytes for in vitro malaria liver stage studies
Step | Problem | Possible Reason | Solution |
|---|---|---|---|
Perfusion | Poor cell dissociation | Poor digestion | Make sure the Digestion solution is at 37°C |
Tissue disruption | Lower down perfusion speed | ||
Cell death | Excessive collagenase digestion | Reduce the time/volume of perfusion with Hepatocyte Liver Digest Medium | |
Cell isolation | Low recovery | To many cells loaded on the gradient | If using more than three lobes, increase the number of gradient tubes |
Gradient disturbance | Setup new gradient, making sure the layers do not mix | ||
Altered gradient densities | Check that the gradient centrifugation temperature is kept at 20°C and Percoll solutions at room temperature |