Hello,
I was wondering if anyone knows of a resource which lists cell lines (common ATCC.org ones) that bind RGD (and ones that don't). Ultimately, we'd like to obtain a cell line that has high expression of the integrin (positive control for RGD binding) and one that has no (or very little) integrin expression (negative control for RGD binding). For example, Hela and HEK293 (but I can't confirm their RGD-binding profiles).
Thanks for your help!
Jim
Hi Jim, most if not all cell lines grown is serum express some aV integrin subunit or RGD binding integrind. Vitronectin is the major adhesion protein in serum and it binds avidly to tissue culture plastic.
Since cell lines are made and propagated in serum containing media, adherent cells are selected for. Non-adherent cells (Jurkat, etc) dont express aV integrins as far as I know.
Hello Steingrimur,
Thanks a lot! It is interesting that Jurkat cells do not since they are propagated in serum too. I should be able to use HeLa for my aV positive, and 293 suspension for my negative. I can try Jurkats as well. I have ordered some FITC-RGD. I will screen some of our cells for binding just to be sure.
Thanks again! Much appreciated!
Jim
Hey Jim, just bear in mind that aV integrins do not bind tightly to RGD sequences. Including 1-3 mM Manganese chloride in your assays will improve binding.
Thanks a lot for that advice! I'll definitely do that! Also, will I be able to trypsinize the adherent cells following the RGD binding (for flow analysis), or will the intregrin get cleaved and I'll lose my signal?
Jim
Hey, it depends on the antibody. If it is a poly then I think there is no worry.
Hi Steingrimur,
There is no antibody used in this study. I just want to bind a fluorescent cyclic RGD peptide to cells. Since the cells are adherent, and I want to quantitate brightness (and subsequently, number of integrins per cell), I need to detach the cells. I was thinking trypsin would cleave the integrin and release my fluorescent RGD peptide from the cell.
Thanks,
Jim
Hmm. Trypsin would be a problem since it cleaves cell surface proteins.
You could use EDTA to detach cells, but integrins are Me+2 dependent and in my hands EDTA is not a very efficient cell dissociation agent.
If EDTA doesn't work for you, you could try attaching the RGD peptides to beads and allow cells to attach to the beads. Isolate the beads and treat with trypsin.
Just keep in mind that fluorescence is not quantitative like absorbance. Absorbance follows Beer's law. Fluorescence does not.
Ah yes that is a good idea. I did realize that fluorescence isn't linear, but it should tell me if a cell line has more than another (if not exactly how much more).
Thanks for the great suggestions!
Jim
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