Hi, I used fibronectin in concentration of 10ug/mL, to dissolve lyophilized powder add sterile water and let equilibrate for 30min. at RT, do not agitate or swirl.

Normaly I coat my plates with Collagen rat tail in conc. 4mg/mL or 5ug/cm2 and I have very good attachment of HUVECs.

Hope I could help

I have coated membranes for permeability assays using vascular endothelial cells with 0.1% gelatin. produced very tight monolayers.

Hi Lisanne,

from your polymer mixture used for the membrane it seems that it is a rather "inert" material. Are you sure fibronectin is actually attached to the membrane? Did you tried any labelled FN/antibody staining to prove that? Without that, you can not tell whether you have a functional membrane or not. This would need to be the first experiment before using cells. From the values you get, it seems that there is no FN attached.

If no FN is attached, think about how to activate the membrane - e.g. plasma treatment. Always run activated, non-FN functionalized membrane as well (since cells attach better to e.g. PTP then on non-plasma treated PS).

Hi Marcus,

Yes I did check with fluorescent labelled FN, the fibronect does attach to the membrane. I also tried NHDF, instead of HUVECs, and for these cells there is a significant improvement, 66 +-6 cells/cm2 (uncoated), to 106 +-2 cells/cm2 (coated)

Also if we check proliferation, after 4 and 7 days, there is a significant difference for both HUVEC and NHDF.

I considered shortly plasma treatment, but since I wanted an easy option and there were so many variables, I decided to go for coating first.

It may be possible that the FN is washed off during culture, HUVECs do not create a lot of their own ECM, so they feel bad then. Fibroblasts are really robust and may do fine with only little FN amount left.

So did you tried to label the FN after you treated the coated membranes same as for cell culture? Maybe there is also something from the medium sticking to the non-coated membrane with time.