I am interested in dissociating small, solid mammary tumours from MMTV-PyMT mice (which aren't necrotic or angiogenic) and using flow cytometry to assess levels of innate and active immune cells within the tumour. Is it necessary to perfuse the mice before dissection to get rid of any blood that may be present?
No necessarily. Once you have excised the tumor, cut it in small pieces, incubate with collagenase at 37C, then gentle dissociate between 2 microscope slides, pass it through an 18G needle, spin down. If there is no blood * resuspend the pellet in your buffer and pass it through the mesh for staining and flow. If you find blood incubate with some hemolytic buffer for 2-3 min @ RT and then proceed from *
Thanks, I have just been dissociating the tumours as I would normally do for isolating epithelial cells then lysing red blood cells in isotonic buffer. I haven't seen any publications with people who perfuse for mammary tumours.
Yes, I was working with NeuT mice and didn't perfuse. That is the protocol I used and gave good results. Good luck!
It really depends on the vascularity of the tumors. We don't typically do this for mammary tumors grown locally. We do perfuse mice if evaluating metastatic lesions in the lung and liver to make sure we are not evaluating cells in the bloodstream that are not infiltrating into the tumors.
Thanks for your feedback! I'll stick with what I'm doing and hopefully can find a few references to back it up in case of publications. I'm not evaluating metastases (at this stage) but I'll keep that in mind, cheers!
Hi Sarah, did you end up identifying a good protocol for isolating immune cells from the tumors of these mice? I'd appreciate it. Thanks!
Hi Colt, basically I just dissociated the tumours as I would for getting out single epithelial cells. However, with epithelial cells, after digestion, I would do a very slow spin to try and get rid of some of the immune cells and also do lineage depletion via magnetic separation, which I didn't do in this case. Processed the tumours as in the literature and in my paper http://www.nature.com/onc/journal/vaop/ncurrent/full/onc201566a.html
Once I had the single cells I just stained them live for immune cell markers and analysed by flow cytometry. Hope this helps! Let me know if you would like more details.
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