Hello everyone,
I have been trying hard to isolate pure primary MLEC to no avail. I always get contamination with fibroblast cells which actually outgrow the endothelial cells. Any recent and better protocol to assist me?
Thank you
You should use two antibodies (first pull with CD-31, passage than Icam-2) and magnetic beads.
You are not alone, Nathaniel. Purification is not the problem. You can easily purify CD31+CD45- cells using flow sorting or immunomagnetic enrichment (miltenyi biotec, initial depletion of CD45+ cells followed by two-three passes through columns for CD31, you can get up to 95% of CD31 positive cells).
The problem is that murine primary pulmonary as well as cardiac endothelial cells have very limited capability to proliferate in vitro. It can be enhanced by the treatment with combinations of growth factors, such as EGF, IGF, VEGF and FGF. However, the cost-effectiveness ratio is low.
One easy way to receive pure population of endothelial cells for the experiment would be to use second enrichment of cells grown in culture. Usually, I use one pass through the column to retain CD31+ cells from the whole organ cell suspension. Then I allow CD31 enriched cells to grow for three to five days. Then I perform second enrichment of cells and use CD31+ cells for the experiment.
Good luck:)
Ruslan Rafikov thank you. Can you please recommend a company we can get ICAM-2? We have tried for the past 4 months from different companies, all to no avail. They say they have stopped producing.
Sergey Ryzhov
Thank you very much for the information.I will try and let you know of the outcome.
Hello ,
Just to add a few words to the above suggestions: you may consider using cell culture medium containing D-Valine, which suppresses fibroblast growth.
Best of luck!
Gediminas Cepinskas
Please what concentration of the D-Valine do you use? Also, can you please provide me with a reference to look at? Thank you.
Hi Nathaniel,
We do not add D-valine by ourself, since the medium has to be specially formulated (e.g. D-Valine has to be substituted for L-Valine). You can purchase this medium from several sources (e.g. Sigma), but we use Endothelial Basal Medium MCDB 131 with D-Valine (Cat #E3000-01D) from USBiological Life Sciences. Hope that helps. Best of luck with your endo cells.
Gedas
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