Abstract
The islets of Langerhans are clusters of endocrine cells located within the pancreas. Insulin-producing beta cells are the major cell type within islets, with glucagon-producing alpha cells and somatostatin-producing delta cells the other major cell types. The beta cells are the target of immune-mediated destruction in type 1 diabetes (Graham et al., 2012). Failure of beta cell function accompanied by loss of beta cell mass is also a feature of type 2 diabetes (Wali et al., 2013). Therefore studying the biology of pancreatic islets is important to understand the pathogenesis of diabetes and to develop new therapies. Here we describe the isolation of mouse islets. This requires gentle enzymatic and mechanical digestion of the exocrine tissue and density gradient separation (Chong et al., 2004; Liu and Shapiro, 1995; Thomas et al., 1998). We then describe how islets can be cultured whole or dispersed into single cells for use in a variety of in vitro and in vivo analyses. Using this protocol reliably results in the isolation of 200-400 islets, depending on the strain of mouse.
Materials and Reagents
Equipment
Procedure
Notes
Recipes
Acknowledgments
This protocol has been developed and refined by members of our laboratory since it was first published (Chong et al., 2004; Liu and Shapiro, 1995; Thomas et al., 1998). We acknowledge all members of the group who have contributed to this process. We routinely utilize this protocol in our studies of the pathogenesis of type 1 and type 2 diabetes in mouse models (Chee et al., 2014; Graham et al., 2011; Quah et al., 2014; Wali et al., 2014; Zhao et al., 2015). This work was funded by a National Health and Medical Research Council of Australia (NHMRC) Program grant (APP1037321) and a fellowship to HET (APP1042735). The St Vincent’s Institute receives support from the Operational Infrastructure Support Scheme of the Government of Victoria.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
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.
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/ml2 x 0.60mg/ml2 x 0.70mg/mlAnd 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 be2 x 0.50mg/ml2 x 0.55mg/ml2 x 0.60mg.mlOne 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