I am attempting isolation of islets from rat pancreas. I will be cutting the tissue (1x1mm) for collagenase digestion instead of purfusing collagenase into the pancreas. I would appreciate any suggestions for maximizing the yield, particularly about collagenase concentration, time of incubation etc.
This method will work, but it will not give you the best yields. I really believe that intraductal collagenase infusion gives the best results. In the rat it should not be possible to do so via the common bile duct, as long as you occlude the duct above. You should observe distention of the entire gland. Collagenase used may be Sigma Collagenase V. Time is approximately 20 minutes. It may vary according to the collagenase. Gradient purification should be on Ficoll/Eurocollins based gradient, or even Albumin/Eurocollins. The latter may be more expensive but minimizes toxicity.
I should correct my answer above: "it SHOULD be possible to do so via the common bile duct..."
Yes I agree that the perfusion method works better..... You have to standardize the duration of digestion for every collagenase batch.....for purification I tried hand picking the islets after passing them through a bigger size mesh (700 i think).. another most important factor is finding the right batch of FBS. I found the isolation easy. Maintaining them was a task.
You can see the duct running from the liver to the duodenum. you can cannulate near the duodenum (and then you can see the pancreas inflate on your right side) or from the liver towards the duodenum (and then it's hard to see the pancrease).
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