Dear all,
I use fibronectin with a concentration of 5µg/ml (diluted with growth medium) to coat plastic wells. The cells always attached, but when I try the same on glass chamber slides, the cells won't attach. I also used a higher concentration (10µg/ml) of fibronectin, but this doesn't seem to work. Incubation of the plates with fibronectin is usually a couple of hours.
Is there something different in coating glass instead of plastic?
Thanks,
Bert Van den Bogerd
Is the plastic you are using plasma-treated or not? 5ug/mL should be more than adequate for FN coating. Are you making or purchasing the glass chamber slides? Maybe there is another factor here such as an adhesive that is having a negative impact on your cells rather than a problem with FN adsorption?
Hi Daniel,
Thanks for your answer! We have glass chamber slides from Lab-Tek (8 well glass slide). Attachment of the cells is always an issue, but it is so strange there is such a major difference (as in no attachment on glass) compared to the plastic.
Kind regards,
Bert
Hi Bert. Do you plate cells in the presence of serum? Do you use any sort of blocking agent after the FN coating step? I've had good success with cell attachment to FN-coated Lab-tek glass bottom dishes, so am surprised that you are experiencing difficulties. Note that plastic will readily adsorb serum proteins and will allow rapid cell attachment even if your FN coating failed. This is also true for glass, but the adsorption of serum proteins to glass is slower.
Could you try coating the glass in FN, then air drying them (without washing of the film of FN)? This will dry fix the FN onto the glass better. You could do this a few times. Dip them into the soltion of FN briefly, air dry, then repeat. Do you have to use FN? You may get better results using gelatin.
Thanks for the answers! I will try them and keep you updated about the outcomes.
Bert
Good luck, Bert. If you are purchasing the FN commercially and continue to have problems, you may also want to test another lot of fibronectin. Occasionally, there can be problems with getting lyophilized FN into solution properly, such that your working FN concentration might be a much lower concentration than expected.
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