Hi,

I do not have a tested protocol but I would suggest to use lethal dose of antibiotic ( on the many classicaly used for clone selection after a vector transfection, like puromycin or geneticin ). After a couple of days most of the cells should be dead and be easily removed by changing the media within the well.

I'm afraid that if you use chemical (alcool for example) you could damage your ECM, or their epitopes (if  you plan to stain the ECM with immunostaining).

Good luck,

Romain

Thanks for that Romain, that might actually work. And yes, I do plan to immuno stain the ECM so important not to damage it in any way.

Thor

I would suggest using the protocol for denuding 3D cell-derived matrices of cells. Here, an extraction buffer consisting of PBS, 0.5% Triton X-100, and 20mM NH4OH are used to decellularize culture dishes, leaving the protein ECM intact. DNAse is also used to remove DNA debris.

Thank you all for your contributions. I'll try the combo of TritonX and ammonium hydroxide. I shall report the results once the data is in.

Hi Thor, do you have any results using the combo protocol? I have also tried it, but I am afraid that I have lost some ECM components.

Dear all, we have tried the protocol with 20mM NH4OH and 0.5% Triton x100, but after staining for DAPI/phalloidin, to our surprise, nuclei were still present, very well preserved. Is there any trick to the protocol? To mention, we are decellularizing cell sheets. If we keep the solution for more than 5 mins, are sheets detach, especially if they are on TCPS. ECM seems to attach better on glass, so we do not lose it when performing washing. 

Hi, I am new to this technique. Wondering how confluent the cells have to be grown (I believe in complete media)? Were the ECM intact and cover the whole well?

Please advice