Has anybody had any experience preparing tough tissues (like gut) for FACS analysis? Other than collagenase digestion, using a cell strainer and removal of insufficiently digested parts by centrifugation, what else can be done? Are there any suggested protocols?
Hi, I have not worked with gut tissue, however my lab does quite a bit of work dissociating basal cells from stratified epidermal tissue and are able to get very high % yields as well as survival for FACS.
Our tissue is dissected out and kept in cold low ca ringers. It is mechanically cut into small bits and trypsin digested in .05% trypsin/edta at 37C until the tissue sediments and balls up. Around 10-15 minutes is enough for it to become visually "sticky" which is what you want.
We take this tissue ball and add an enzyme cocktail which contains collagenase, hyluronidase, trypsin inhibitor, papain, and DNAse made up in regular ringers.
This mixture is incubated at 37 with gentle rocking for 15 min. The sup is decanted and placed on ice, while another aliquot of enzyme cocktail is incubated with the tissue again for another 15 min.
Pool the sup and filter through 120 micron mesh. Pellet at low speed and resuspend in low cal ringers. Pellet again and resuspend with HBSS/EDTA. Filter through 35 micron mesh, pellet and resuspend in a volume for either directly FACS or for staining.
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To address some issues you may see here:
The >15 min trypsin digest: we have done tests and cannot see a difference in FACS of cell surface receptors. It appears that with all of the good ECM to chew up, receptors aren't really affected. It may pay for you to do some tests in your tissue though. The trypsin digest is essential in our tissue to retrieve basal cells.
DNAse in the enzyme cocktail: i have no idea why we have it...but we've never gone without it. So it could be one of those reagents that we use but don't need it, but we've gotten our protocol down so well we don't want to change it.
If you would like, I can provide details on the reagents, I just can't remember them at the moment :p
Good luck with your facs!
Dear Brian, thanks a lot for this excellent answer! You gave me very valuable suggestions!
Meanwhile I found ome papers where enteroendokrine cells have been isolated for FACS. If required, I can post links to the papers.
Best, Bernhard
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