In general I prefer short vs long, but you will have to try it on your tissue before (how short it can be, sometimes even 10 mn is enough !).

I would also strongly recommend a manual / automatic tissue disruption before collagenase (either chop tissue with razor blade or use gentle MACS is you can access one). This allow better access of collagenase to tissue and digest it better and faster.

Collagenase work better when done under gentle agitation (agitator 200 rpm or magnet and stirrer).

Good luck !

I agree with Juliette and Holger that long incubations in collagenase can certainly alter your F4/80 staining for FACS. I have performed immune cell profiling in colon tissue and observed a number of cell surface markers were altered by collagen and/or dispase. We came across a publication that describes some of these (Autengruber A, Gereke M, Hansen G, Hennig C, Bruder D. Impact of enzymatic tissue disintegration on the level of surface molecule expression and immune cell function. European Journal of Microbiology & Immunology. 2012;2(2):112-120. doi:10.1556/EuJMI.2.2012.2.3.). For our purposes, we chop the tissue using a razor blade, then digest in 200U/mL Collagenase Type I for 1 hour (also with DNaseI and FBS). Following digestion, we run the sample through a 40um cell strainer using the plunger from a 5mL syringe to further smash the tissue through the strainer.

Dear Alyssa

I don't have any experience with mammary glands but I have to work with different others tissue disaggregation (from cancer lesions, healthy skin, healthy gingiva, from lipossuction). We also search leukocytes from those tissues but we never have worked with so long times of incubation with colagenase.  I've sent some articles for you. I hope they help you.

Good luck

Hi,

I would prefer to use Liberase TM (2mg/ml) at 1:5 diln for  30 min and then block the reaction by adding 100mM EDTA. Otherwise, treating with collagenase (2mg/ml, Roche) is also fine, but treating overnight is too long. However, u haven't mentioned the concentration of liberase or collagenase u r using for digesting mammary glands. I would prefer u to follow the concentration of enzyme mentioned by Jessica in the above post and u can put chopped tissue in the  solution and incubate at 37C for 60 min. After incubation add EDTA and then pass it through the filter of interest. I would also suggest to use series of filters before enriching or staining ur samples. As mentioned by Juliette and Holger, u can use Automacs, which is really good at isolating cells from different tissues .

Best,

Atif

Hello everyone. I stopped getting notifications about responses for some reason, so I didn't realize so many people had replied. The protocol that I'm using was inherited from a post-doc and uses Liberase TM at a concentration of 0.5 mg/mL. The Liberase is diluted 1:10 for the digestion for a final conc of 0.05 mg/mL. The tissue is minced before digestion and digestion typically takes 30 mins to 1 hr. From there I strain through a 100 um filter and do a gentle spin at 200xg for 10 mins, since I am also collected adipocytes. Another option that has been offered is to do mechanical dissociation in a Stomacher, and skip enzymatic digestion altogether. Our flow core manager mentioned that some people in the flow community believe that mechanical dissociation can activate macrophages. This last statement is hearsay and I have no evidence to support it, I just think it's interesting.