Surprised that TE doesn't work.  3T3 ECM is mainly fibronectin, so TE should release your cells.  Did you rinse the plates with PBS or serum free media once or twice before adding the TE to remove competing protein such as serum?  Also, check that the TE is active and concentrations are correct.

sorry Robert, I wasn't specific enough, I need to detach only my cos-7 cells, if I add trypsin, I detach the cos- 7 but also my matrix, which I don't want to.

What are you trying to study and what do you intend to do with the detached the cells?   Do you also want to preserve the matrix on the culture dish?  

These factors will determine what methods may work for detaching the cells.

Hi, Despoina!

Have you tried scratching them? I don´t know if this will release the matrix too, but I think it may be useful....

As Robert said, you must know what is the intended use for your cells and matrix.

Hope it helps...

Hi guys, thanks for your answers! So I don't want to detach my matrix along with my cells, so I am not sure if scratching would be good! Ideally I want to detach them, lyse them and probe for several proteins.

I am not sure you can do what you want.

I think the matrix is so intimately involved with the cells that you will need to disrupt it to get the cells released. You could just try EDTA alone? Or you could pop the cells with  hypotonic shock in situ?

Also I am not sure why you want to avoid the ECM at this point. If you want the cellular proteins then these will be detectable upon lysis anyway and the signalling won't be affected by co-purified ECM.

Maybe I haven't understood your assay requirements sufficiently.

Best wishes.

Jon

Hi Jonathan,

thank you for your answer. I will try EDTA and the hypotonic treatment, maybe it works. I also read about a solution called accumax that claims to detach cells from 3D cultures, have you perhaps used that? 

I don't want the ECM because it will contain proteins on its own and I want to see what types of proteins will be produced by my own cells, I mean I want to see what is in the intracellular part, not anything secreted. 

Thanks for your help!

You might want to try EGTA instead of EDTA.  If the cell-matrix binding is Ca++ dependent, then EGTA may work better.  For some cell types, EGTA without trypsin is sufficient to detach the cells.

Hi Despoina,

I've done some WB experiments on cells plated on matrices. To lyse them and preserve the matrix I normally use an extraction buffer made of 20mM NH4OH and 0.5% Triton X-100. In my hands, ECM proteins are not present when you use this buffer. Hope this helps, let me know if you need more info.

Good luck

Elena