Dear Romina, here are some tips that hopefully will help you in titrating the collagenase.
If you are using Roche Collagenase P you need to contact them to try and get a data sheet on the batch of Coll P you are testing. They do not send this out anymore as they are apparently making the activity at 1.6 Units as a standard now. However, the trypsin is always different, so you need to make sure that the trypsin level isn't too high.
Ideally trypsin should be between 1-2u/mg Lyo. If it is too high ie. up around 4 like some of the batches we have tested the islets over digest and we get horrible islets with low yields. Our current batch is 1.870 u/mg Lyo.
So given the activity levels now and if your trypsin isn't too high you should be using somewhere between 0.5 and 0.6mg/ml and between 15-18 minutes digestion time. As for the actual set up for optimization this is what we would normally do:
6 mice normally young 6-8week old NOD males.
2 x 0.5mg/ml
2 x 0.60mg/ml
2 x 0.70mg/ml
And do one of each concentration for 15min and the other 18min. At the end make a comparison and then refine for your next experiment. For example, if the best islets were in the 0.5 and 0.6mg/ml groups and looked better at 15 minutes the next try would be
2 x 0.50mg/ml
2 x 0.55mg/ml
2 x 0.60mg.ml
One of each concentration for 15min and the other 16mins. It would normally take 3 or 4 goes refining it each time before we would be confident we are getting the best quality and number we could.
I hope this is helpful, please email me if you want more information.
kind regards,
Helen
2019-01-04 20:02:01