I want to isolate relatively pure populations of mouse uterine epithelial cells for western blot and gene expression analysis. Older published protocols use 1% trypsin + dispase or other additional enzymes, at 4 degrees for an hour, then RT for up to an hour, and 37 degrees for 10 min. I will try this approach but am concerned about changes in the cells over those extended times.
I would also be interested in any methods for purification of endometrial stem cells.
Protein and mRNA composition could be studied over the course of these conditions and time points, but are most often not available. Having said that, major changes would not be expected at the protein level, but at the mRNA level it is a WHOLE different ballgame, where MAJOR changes would be expected.
you can use our protocol for isolation of human endometrial stem cells in our papers. I think this protocol is suitable for isolation of mouse uterus cells.
http://www.ncbi.nlm.nih.gov/pubmed/23338937
Perhaps I should back up - if I have a mouse with a epithelial specific knockout, I was assuming I should try to isolate pure epithelia for western/gene expression... But maybe it is sufficient to lyse the entire uterus (using mortar and pestle, for example) rather than worrying about separation of epithelium from stromal tissues? Can anyone describe what is standardly done in the field? Again, this is a new area of interest for me.
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