Recently, I am trying to isolate islets from db/db mice. I use the same protocol I regularly use for islets isolation from mice but I can't get any islets or very few pale islets attached to exocrine tissue which I can't use for my experiment. Is there a special consideration during islets isolation from db/db mice?
Normally standard procedure works fine with some tweaks (adjusting the time as one watches the digestion progress)... but you have not included one very critical piece of information - age your db/db mice. If they are old enough to be diabetic, your results are entirely what one might expect.
That is about the average age when db/db mice become diabetic. Your results are not surprising.
Did you check blood glucose levels of your mice the day of islet preparation?
Dear Dina,
We isolate islets from db/db mice regularly and your issue sounds right in line with what often see. The islets from diabetic mice are often very pale (degranulated), rough around the edges, and slow to recover from the digestion process. Your mice are almost certainly diabetic at 16 weeks of age; Jackson Lab reports diabetes onset around 4-8 weeks of age, which is what we see as well.
That said, you ought to be able to retrieve about half of the normal yield you would get from an age-matched control without any change to your protocol. We don't typically go out beyond 12-14 weeks of age for our studies, but we are still able to get 150-250 islets (vs. 300-500 for a heterozygous or wt mouse).
Are you using a gradient? Assuming you use a gradient to separate islets from exocrine tissue, my best suggestion would be to skip that step next time. As soon as the digestion and wash steps are over, dilute the pancreatic prep 1:10 in whatever buffer solution you normally use and keep this mixture on ice. Take a sample of this pancreatic digestion and take a look under the microscope. You will either find broken apart pale islets within this sea of exocrine tissue (possible overdigestion), islets that are mostly embedded in exocrine tissue (possible underdigestion), or a lot of decent looking islets that are somehow being lost during your gradient step. It is slow and pain-staking, but you could handpick the islets under the microscope. You could always go back to the gradient step once you figure out what needs to change in your protocol to improve the yield.
Best of luck!
Dear Dr. Craig,
Thanks for your very informative answer. yes I use the gradient method. On the same day I extracted the islets, I checked under the microscope i couldn't see them so I desperately left them in culture for two days and I checked them and could see some with rough edges and some embedded in exocrine tissue. Next time I am doing the isolation, I will follow your advise.
Onset of diabetes can vary, in your case when do you observe diabetes?
if you are interested in islets that have substantially or completely lost beta cells, beginning with mice at 6-8 weeks after onset of diabetes is a good choice.
While you are shaking pancreas after inflating it with proteases in situ, rather than going to your normal time, check a small drop of the mixture under a microscope quickly for the progress of digestion - once every minute or so. After a little experience, you will know the gap between the degree of undergistion in the mixture on the first step that corresponds to relatively clean islets at the end - this is what I had alluded to earlier by "tweak".
Well pst onset of diabetes, the "islets" look rough and different is largely a consequence of destruction of beta cells, not merely degranulation. One can confirm the effect of degranulation on appearance of islets by looking at db/db pancreas - they are larger than normal (owing to compensatory hypertrophy and hyperplasia), relatively smooth and intact shape, degranulated but producing large amounts of insulin, as evident from their in situ insulin profile.
Best,
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