Hi,
I isolated cardiac fibroblasts from a mouse and cultured them in P.1.
After mRNA extraction I saw high expression levels of CD146 gene, so I wonder if at that point it's possible that ECs (endothelial cells) were also present in the culture, as this is an endothelial-characterizing marker.
The isolation protocol which I use for primary fibroblasts culture does not include any particular stages for the removal of ECs, but for what I know ECs should be excluded after going through trypsinization (cells are in their first passage), and fibroblasts tend to take over.
Last time I checked CD146 gene expression - cells were in P.3 and did not express CD146..
Thank you very much in advance.
Hadas
Hello Hadas,
Wash the cells after 2 and half hours of isolation. It will avoid the attachment of endothelial cells to a great extent. Even if you find one or two colonies of endothelial cells, it will easily go away after the first passage.
the problem is that the isolated fibroblasts only adhere to the surface 6 hours approximately after seeding, and even than not all of them do so, so by washing the culture 2.5 hrs post-seeding I will be losing a great number of cells. most of them actually.
thank you though..
2. 5 hrs post-seeding is sufficient for fibroblasts to attach. We always isolate fibroblasts ( from lung and heart) and get a good yield. Make sure that the tissue is not over-digesting. Excessive treatment with proteolytic enzymes— like trypsin will affect the cells and take a long time to get attached.
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