ECM/Laminin coating and primary cultures

[mophlrkb]

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.

[CrowSan]

Hi it does not seem that you are doing anything wrong (Laminin as you mention needs to be defrosted slowly).
I usually make upo the laminin at 20 ug/ml in DMEM/F12 (4oC) and filter through 0.2 u filter to sterilse.
I add the laminin to the flask and place at 37oC for 3 hours to allow the laminin to coat the plastic (how long do you treat?).
After this time remove the laminin and (importantly) rinse the flask with media to remove any unbound Laminin (other wise it can stick to the cells and prevent them binidng). If not adding cells straight away add media an place at 37oC.
Note: When adding media, washes etc then add this to the side of the flask and not directly onto the laminin/cell surface.
Can erythrocytes stick to non-laminin coated plastic? If so this is not really a good internal control for Laminin treatment. If problems persist then indeed change to poly-lysine.
hope this helps and sorry about delay in response
CrSn