Hello!
I am attempting to analyze cell populations in mouse lung homogenate using flow cytometry. I have seen a variety of protocols that use a range of collagenase and DNAse concentrations, brands, and types (e.g. type I v type IV). Is there no consensus?
Why does the brand or type of collagnease matter? I want to make sure I get consistent single cell suspensions for flow and I'm hoping to purchase these items soon.
Thank you,
Hi Gregory,
the brand of the Collagenase probably does not matter much, but the type actually matters a lot. Different types of Collagenase are all called "Collagenase", but they differ in how pure they are. For example Collagenase Type 1 still contains a variety of other proteolytic enzymes, while Type 4 has the lowest tryptic activity.
The best choice for your prep depends on which cell types you would like to analyze. If you only want to get immune cells and are worried about loss of surface markers (which can be digested off), I would go for a rather pure Collagenase. In contrast, if you would like to isolate some kind of stromal cell that is harder to get out of the tissue, you might need to go with a harsher collagenase containing some other proteases.
There is actually a similar question here:
https://www.researchgate.net/post/What_is_the_difference_between_collagenase_type_I_and_type_IV
Good luck with your experiments!
Florian
Thank you Florian! That was very useful information and now I can test if the 1998 type IV collagenase in the lab still works...ha!
Best,
Also, why is the DNAse added? I see some groups add the enzyme and some do not.
That is handled differently by different labs, but I would definitely recommend adding DNAse. Again, that depends a bit on the tissue that you are digesting and how many cells will die throughout the process. Dying cells will release DNA, and this will actually cause the suspension to become viscous and might cause further cells to die. Therefore, addition of DNAse is recommended.
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