How does fibronectin "crash out" of solution?
Posted 15 November 2012 - 11:20 PM
And how do you thaw out fibronectin stocks (stored at -20 oC)? Millipore suggests slow thaw @37oC, Sigma suggests slow thaw @2-4 oC. Thoughts?
Posted 18 November 2012 - 06:59 AM
I have sucessfully used fibronectin for BAEC cells and resuspend in sterile ultra-pure water, aliquot according to predicted usage, into 1.5 ml tubes and place those tubes into 50 ml conical tubes, cap tightly then store at -20C. I thaw aliquots at 4C, then equillibrate to room temp for a few minutes. Gently invert the tube to mix, never vortex or shake vigorously.
I store the thawed aliquot at 4C if I will use it in the next week, otherwise re-freeze. Avoid repeated freeze/thaw by aliquoting small volumes, you can always thaw more tubes if you need them.
I dilute the fibronectin in PBS to desired concentration and coat with minimal amount, just enough to cover the surface uniformily with a thin layer of solution, incubate at 37C for 1 hour minimum, aspirate the excess just prior to use, wash with PBS, and add cells suspended in medium.
Also, if I know that I need more coated dishes in the next week or two, I coat additional flasks/plates, incubate and wash as above, store in a sterile bag (save sleeves from sterile plates that you've only opened in the hood), seal tightly and place in 4C for up to 2 weeks. I like to leave a thin film of PBS on the culture dish so that they don't dry out in the fridge.
As you know, fibronectin is very expensive and it doesn't go very far. I have been able to reduce our usage dramatically by testing different coating concentrations. Give it a try unless it's already been pre-determined by other lab peoples. The only draw back to reduced coating concentration was slower cell growth, we decided that the cost benefits were well worth the extra day or two waiting for cells to reach desired confluency and plan accordingly.
Edited by Denny, 18 November 2012 - 07:06 AM.