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-   -   ECM/Laminin coating and primary cultures (http://www.molecularstation.com/forum/cell-biology-cell-culture/85848-ecm-laminin-coating-primary-cultures.html)

mophlrkb 06-05-2012 06:44 PM

ECM/Laminin coating and primary cultures
 
Hi all
First time having to post on such a forum and would appreciate your valued input. Am culturing primary choroidal epithelial cell from porcine brains.
We are successfully able to isolate the tissue and pellet/resuspend the cells from established protocols and can visualise the cobble-stone appearance of the choroidal epithelial cells, so know they are there in the culture flask.

Plastic ware should be coated in Laminin, which I've made up in sterile water at 25ug/mL and use 2mLs per T25. The problem is that the CP cells don't adhere but rather float off and are discarded when I change the media on day 1 (post seeding)--to remove unwanted cells. There is something that has attached to the flask/laminin which I think are the erythrocytes.

Any ideas what going on? The media used is DMEM:F12 with supplements as established in various protocols.

I may have made ONE mistake in the laminin coating, removing laminin vial from -20 and transferring to ice-bucket before defrosting at RT, which I'm guessing could have gelled the laminin. I should have gone: -20, 2-8 and then slowly to RT. However, the erythrocytes (or whatever they are) are stuck in the flask.
Was also thinking of trying with poly-l-lysine.

Anyway. Any advice as to other potential causes would be gratefully appreciated.


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