I'm trying to optimize colonic macrophage isolation, and am having trouble wrapping my head around the digestion process. Based on published methods, there are usually two steps involved in digesting murine intestinal tissue:

1) Usually involving EDTA, this step removes the intestinal epithelium and intestinal epithelial lymphocytes.

2) Using dispase/collagenase, this step is supposed to digest the underlying mucosa. There is a subsequent filtration through 40uM cell strainer.

My concern is that the second enzymatic digestion and subsequent cell collection is collecting everything. Aren't the aims of these protocols to isolate only the mucosal cells, not the underlying cells within the submucosa and muscle layers? Does this enzymatic digestion remove the mucosal layer, leaving it suspended in the media (with the remaining submucosal layers sinking to the bottom)?

Below are a couple examples of these protocols. Thanks very much for any advice you can provide!

https://www.jove.com/video/4040/isolation-characterization-dendritic-cells-macrophages-from-mouse

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4913542/pdf/nihms795234.pdf

https://www.jove.com/video/54114/isolation-flow-cytometric-characterization-murine-small-intestinal

Cheers,

Scott