Hi,
After shacking the mice colon mucosa in HBSS with EDTA, DTT at 37oC for 20 min, then sediment for 15 min at 37oC, then I can get the crypt epithelium.
But how to get the single cell suspension form above crypt epithelium for further analysis, such as flow cytomertry, cell culture? Do I need to use trypsin, will trypsin destroy the epitope of the protein when I do staining? Do I need to use the digestion enzyme to get single cell suspension? Or is there any simple method? Many thanks!
Xin
check this paper in nature protocols, it has all the details and works great for human and mice.
Article In vitro expansion and genetic modification of gastrointesti...
Thanks for your message! But I can not get access to the publication.
Another question is that :
When I treat the colon with EDTA and DTT to get the crypt epithelium, is the intraepithelial T lymphocytes connected together with the crypt epithelium, or the intraepithelial T lymphocytes already become the single cell suspension. I am now need to collect the intraepithelial T lymphocytes, I wonder if I only collect crypt epithelium,and digest them, will I loss the information on intraepithelial T lymphocytes.
Thanks!
Xin
I'm not sure what the yield will be. You certainly will get ILCs in your cell prep that way though. You don't need the isolate crypts alone for your ILCs, just get the epithelium eg using EDTA followed by other enrichment/purification methods.
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