I'm struggeling to get lymphocytes out of gut tissue (inflamed small intestine). We have been using different percoll gradients and protocols, but nothing worked out. Would be great if anyone could help! Thanks a lot!!
Hi Henrike,
I did this with gut tissue of pigs, see file, regarding isolation Peyers Patches, hope It works also for you,
Good luck
Peter van Kooten
1.You can use mechanical and enzymatically degradation protocols firstly.
2. Ficoll density gradient can be applicable.
3. FACS for purified lymphocyte .
Hi Henrike,
Isolation of lymphocytes from the small intestine lamina propria should be fairly easy. I think the most important caveats are to start with properly cleaned tissue (fat and Peyers patches removed) and to have an appropriate enzyme mix for digestion.
I can recommend the following protocol (from our lab):
Isolation of T cells from the gut
Reißig S, Hackenbruch C, Hövelmeyer N
Methods Mol Biol. 2014;1193:21-5
If you encounter any problems with this protocol please don't hesitate to contact me!
Greetings from Mainz :)
Tommy
I don't have much to add to the previous answers. Only that including an anti-CD45-APC (or what ever fluorochrome fits in your scheme) and gating on the CD45+ cells will GREATLY clean up the rest of your analysis. Also, adding DNase I (Roche, 10 mg/ml) to your enzymatic mixture helps the "snotty" mess that comes out. Dead cells along with the mucous can make the prep quite "gloopy".
Hi Tommy and Paula, many thanks for the protocol, I'm gonna follow this next time! :)
Hi,
Separate gut tissue and wash with PBS. Then dissect and then digest with Collagenase 1mg/ml, DNAse 50 mic/ml, Hyaloronidase for 45 min at 37 degree C and after pipeting the sample should pass through cell 100 mic mesh and lymphocyte seperated using ficole method.
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