Hi, I failed to isolate primary murine intestinal epithelial...

Yes, it was murine small intestine. The crypts started to die after day 4 and they were not overcrowded. We were abled to observe few budding point when we used 20% R-spondin condition medium. We did exchange the medium but the crypts could not survive. We did check the Noggin and it met the criteria as well. After reconstitution, we kept in small volume and stored at -80 degree celsius meanwhile thawed solution kept at 4 deg celsius. Polyps culture able to growth with the same batch of Noggin and mEGF. We will check R-spondin 1 activity again for double confirmation.

This was murine small intestine (SI), right? Because cultures derived from human SI require additional growth factors. Generally, your crypt isolation seems to me very successful, because you observed enterospheres on day 1. However, it seems that there are inconsistencies with the culture conditions. At what day crypts started to die? Were the crypts overcrowded? (how many crypts did you observe in one well on day 1?) Did you exchange medium when the medium turned yellow, and observed if at least some crypts can survive? If we exclude bacterial/fungal contamination, then probably one of the growth factors was not working optimally. Did you add Noggin to the culture media? How do you store R-Spondin conditioned medium and recombinant proteins after reconstitution?

The crypts were isolated from small intestine and no contamination observed. For troubleshooting purpose, we used 20% R-spondin condition medium and it was in yellow color when the crypts started dying. We used recombinant R-spondin 1 and Wnt signaling checked using TOP flash assay which met the criteria.

Hi Sau-Yee Kok, thank for your interest in the protocol that we published and for your question. Did you isolate crypts from small intestine or colon? Did you check if cultures are not contaminated with bacteria? Was the medium pink or yellow when crypts started to die? What was the source of R-Spondin? Did you use recombinant R-Spondin?