Does anyone know of a method/tip to avoid interstitial cell contamination from endothelial cells? I have been trying to isolate valvular endothelial cells (pig) and keep getting interstitial cell contamination.
Any help would be greatly appreciated!
Do you see this contamination right away or after a passage or two? Could you try MACs or FACs sorting using PECAM or VEcad either after your initial harvest or after your founding passage, and replate a purified population...... maybe trying different digest times to help elimante excess cells "tagging along" with the ECs"
https://www.ncbi.nlm.nih.gov/pubmed/19137801
Thanks for sharing this paper! Yes, I usually see the contamination few days after the isolation when the cells starts ti grow.
I haven't tried the other techniques using antibodies because I am planning to use these cells for long period of experiments. I am afraid that it will alter my results when I do gene expression.
You can try culturing on matrigel. Thus you can enrich the epithelial cells in your case the endothelium.
MACs/FACs sorting shouldn't hurt genetic expression in your cells ....we MAC/FACs both primary and cell lines and culture them just as well as non-sorted cells. On the other hand, Gene expression changes will occur as an effect of in vitro culture no matter what, epsecially with passaging (certainly with endothelial cells as they can enter replicative senescence, I had to study that at one point), so I wouldn't worry too much about changes from sorting, if anything, the purified population and matrigel would enhance endothelial growth. If purity is a problem due to meager starting number (mouse endothelial can be difficult to get in large numbers sometimes) you might be able to try "pooling" littermate clones and redefine your "n" as number of experiments rather than number of animals????
https://www.ncbi.nlm.nih.gov/pubmed/21273559
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