I am trying to collect macrophages from the mouse blood, but till now I am unable to do this perfectly. I feel following problems and need some recommendations:
1) Blood in mouse is very less, hardly 250-500 ul.
2) RBSs are easily removed, but it is hard to separated the other cells. I am using the following protocol:
http://www.macrophages.com/isolation-peripheral-blood-mononuclear-cells-pbmcs-whole-blood-and-subsequent-culture-macrophage-cel
3) I don't know what percentage of Ficoll-400 is suitable. Sigma Aldrich is recommending 50%, but it is not working well.
4) Should I directly start the macrophage separation from mouse blood after collecting the blood, or may I store the blood for some time?
5) What temperature and how much time is suitable for storing the mouse blood?
Please share your expertise. Thanks.
Hi,
You should not need to use Ficoll for mouse blood, instead we just ensure RBC lysis is complete - we use Biolegend RBC lysis buffer, but any should do. Following this block FC receptors with anti-CD16/32. Then stain with anti CD115, APC works well for me. If you have the option to flow sort, then you can further separate these cells on Ly6C surface expression (I would recommend a lineage gate to remove T cells, B-cells and granulocytes). If not, then you could use an anti-APC bead selection kit to pull out your monocytes (for example http://www.stemcell.com/en/Products/All-Products/EasySep-Mouse-APC-Positive-Selection-Kit.aspx).
I typically only prepare blood monocytes for flow cytometry, but others in our lab have cultured them using this approach. You should be able to get ~100-200k cells from 500ul mouse blood, which will be a roughly 60:40 split between the Ly6Chi and Ly6Clow monocyte subsets. In culture these cells will rapidly differentiate into macrophages.
You really need to process the blood as quickly as possible - I can't stress enough that fast processing is essential for good cell numbers. Work on ice too.
Hello Mr. Salman Ul Islam ,
From my experience of using FIcoll , Lympholyte and ACK lysis buffer , i will recommend you to use ACK lysis buffer as it's the best method to isolate the respected WBC. I have isolated macrophage by all of these techniques and i can help you, if you need.
Feel free to knock me - [email protected]
Best regards,
Babu
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