Hi All,
Immunology novice here, looking for some advice on mouse monocytes.
We are looking to isolate mouse monocytes suitable to demonstrate In vitro ADCC. we tried out two methods:
Mothod 1: MACS negative selection kit for mouse bone marrow
This method yielded ~70% pure CD11B+Ly6C+cells. the remaining 30% seems to be seems to be CD11b-Ly6C+ positive. We have markers on our panel for lymphocytes, and neutrophils and it was all negative. Could this be some kind of precursor cell?
In addition, this kit seems to use Fc block (this could be a problem for ADCC) when incubating with the antibody cocktail. We attempted to isolate them without the Fc block reagent. Of course our yield was reduced and the purity (CD11B+Ly6C+) dropped to 65 % when isolating without Fc block.
Method 2: Involved culturing BM cells for 5 days in the presence of M-CSF and working with the suspension cells or "mature monocytes" based on the following paper:
Article Isolation and Intravenous Injection of Murine Bone Marrow De...
This method yielded me CD115+F4/80+ cells at 87%. How ever, only 30% of these cells express ly6C. and 75% of them express CD206. Wth this method, we were only looking for mature monocytes and not macrophages.
Questions:
1) Which of the two phenotypes would be suitable for ADCC?
2)Do I work with or with Fc block in this context?
2) With the second method, would it help pick up the suspension cells at an earlier time? will that help catch the cells before CD206 expression?
Thank you for taking the time to read this. I look forward to hearing back!
I used to work with BMDMs for my Leishmania model. I checked and there used to be atleast 80% purity of CD11b+ macrophages. I must confess I did not use Ly6c as a marker since as I associated Ly6c+ as a hallmark for inflammatory monocytes/macrophages. Since, Fc block would hamper your overall goal of doing experiments with ADCC I would suggest go for the second method. Also with 5 days time you will find less and less suspended cells which is due to that other cells that may infiltrate (if any) your BMDMs wither away without specific cytokine stimulation. Well of course with stimulation going on you cannot change media. If you gate BMDMs via CD115+F4/80+ a considerable number would be Ly6c- indicating the resident macrophages of the bone marrow. I am not aware your gating strategies but the safest way to identify purity is by CD11b+ for BMDMs to avoid confusion with other cell populations residing in the bone marrow.