Hey there,
I've recently worked on a method for intestinal epithelial cell (IEC) isolation and digestion into single cells (followed by flow cytometry) that is very satisfactory (for my needs), except for one problem: in many cases - usually right after digestion into single cells with Trypsin/TrypLE (alone or in combination with several other enzymes) - I find myself dealing with a very thick suspension that, I believe, is thick due to being full of mucus (it really seems to have the consistency and appearance of snot [nasal mucus]...). That creates major problems. So, I'm looking for a way to remove (or significantly reduce) the mucus, but without removing/killing a substantial fraction of the epithelium. I have tried to use DTT (1.5 mM-5 mM) on ice (before getting to the digestion step), but that didn't help.
Any suggestions? :)
Hi,
Quick question, are you sure the thick substance is mucus and not release DNA/ genetic materials from dead and or dying cells due to the trypsin that you used. Because of your description of it as "snot", I strongly suspect that it is DNA.
Have you included DNase in your digestion buffer (you might have to try it at different concentrations)? If the mucus-like event that you observe is indeed DNA released from the dying cells, then the DNase should take care of it.
Best of luck.
Hadijat M. Makinde Thanks a lot for your useful response!
I am not sure it's mucus. Mucus was my number one suspect, but DNA was my "runner-up" suspect (although I guess it was a distant second in my uninformed mind).
Now, in some of my attempts I did include DNase I (at 200 U/mL [0.5 mg/mL]) and nevertheless had the same problem with many of the samples, but, in part due to your answer, I will now try again with DNase, probably at higher concentration (I saw some papers where it's used at 2,000 U/mL, so I guess that's a concentration I'll try). As I said, it does sound reasonable to me that extracellular DNA causes that issue, even though I thought mucus is more likely to be the cause.
I have no really drastically different suggestions than from what you are doing anyway. But for our gut preps, right after taking the guts out and opening them, we wash our samples twice for 10 min each in 37C warm RPMI+5%FCS+5mM DTT under constant agitation. And this works in my hands pretty well to remove mucus.