We are still standardizing the amount of collagenase. It is...

RB
Romina Bertinat
Dec 15, 2018
protocol Protocol: Isolation and Culture of the Islets of Langerhans from Mouse Pancreas
We are still standardizing the amount of collagenase. It is not a problem of the protocol, is ours!
answer Answer
recommend recommend recommend recommend Recommend
follow follow follow follow Follow
share share Share
2 answers
Sort by: most helpful / most recent

Helen E Thomas Author Answered Jan 4, 2019

Immunology and Diabetes Unit, St. Vincent’s Institute, Australia

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
0 helpful
0 unhelpful
comment 0 comments down up
share Share

Tatiana Jofra Answered Dec 17, 2018

Diabetes Research Institute (DRI) , IRCCS San Raffaele Scientific Institute, Italy, Italy,

It is difficult to standardize the amount of collagenase to be used, depending on the strain of donor mice and especially on the collagenase lot. Before starting a new experiment, it would be useful and important to do a titration if the batch of collagenase was not used before. Starting from a concentration twice greater than two times less than the protocol.
It would be also useful to do the same thing with the streptozotocin.
I hope I have been helpful.
0 helpful
0 unhelpful
comment 0 comments down up
share Share

My answer

Write your answer...

References (optional)

add Add more

post Post an Answer