I am planning to establish primary endothelial cell culture from HUVEC and then to treat them with Dengue virus infected serum for further experiment. I need to confirm my cultured cell as endothelial cell before proceed to my main research.
It really depends on what you want to confirm and how precise you want to be.
Classic EC markers include CD31 (PECAM-1), CD54 (ICAM-1), von Willebrand Factor (vWF), and VE-cadherin.
You could also do all sorts of functional assays, such as looking for nitric oxide production, uptake of a fluorescently labeled acetylated LDL or the presence of the endothelial surface layer (ESL), including glycocalyx, by using a fluorescently labeled lectin, like wheat germ agglutin.
The latter could be particularly important from the standpoint of virus-cell interactions. You could further examine the ESL by staining for HSPGs (heparin sulfate proteoglycans) such as syndecans and glypicans.
It really depends on what you want to confirm and how precise you want to be.
Classic EC markers include CD31 (PECAM-1), CD54 (ICAM-1), von Willebrand Factor (vWF), and VE-cadherin.
You could also do all sorts of functional assays, such as looking for nitric oxide production, uptake of a fluorescently labeled acetylated LDL or the presence of the endothelial surface layer (ESL), including glycocalyx, by using a fluorescently labeled lectin, like wheat germ agglutin.
The latter could be particularly important from the standpoint of virus-cell interactions. You could further examine the ESL by staining for HSPGs (heparin sulfate proteoglycans) such as syndecans and glypicans.
Just to add what peter said, also pay attention to the shape changes with passage. Primary cells change their shape with passage.
Hi
You should also take care about cumulative population doubling (less than 15 - passage 7-8) and have HUVECs in lower passages, because in higher passages they lose markers which are typical for ensdothelial cells (so this may be reason why you get negative results on markers which Peter mentioned). I would recommend to use HUVECs passage 5 (cPD 9-10) for final experiment. And always use for experiments cells with the same age.
Also if you did not work with endothelial cells before you should take care about the confluency, never let the cells become confluent, always passage or use the cells at 80-90% confluency (if you dont perform adhesion/transmigration assays) because after reaching confluency they inhibit each other.
hope this helps a little
Thank you all. Your advises will be very helpful for me as i am doing it for the first time.
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