We are currently planning experiments with the monocytic cell line Mono-Mac 1, which we will be using for HIV infections and analysis of different signaling pathways. As we are using them as a proxy for human monocyte-derived macrophages, we will be differentiating them into a more macrophage phenotype with LPS. However, several of the signaling pathways we are looking at are activated by LPS on their own, and I am worried that the addition of LPS will prevent us from seeing what we would expect. Is there a protocol in which the cells are rested (i.e. non-differentiation media is added) for a period of time following differentiation? I have seen protocols for this with THP-1 cells, but the literature using Mono-Mac 1 is severely lacking.
Dear Emily
LPS both activates the cells and induces maturation. I suggest you rather use a differentiation agent like Vitamin D3 at a dose of 50 nM for 5 days. A paper describing the details is enclosed (Marion Lip VD3).
You may also want to try infection of the sister cell line Mono Mac 6, which has been used sucessfully for this purpose (Lage-Stehr Mono Mac6 HIV). This line is more mature that Mono Mac 1, but may be additional maturation with VD3 can further improve infection.
Best of luck
Loems
While I havent used this exact monocyte line, PMA is the most common agent to differentiate monocyte cell lines into an 'M0' phenotype. With low enough concentrations a pro-inflammatory response can be largely avoided.
With the PMA protocol it is common to replace PMA-media after 48 hours, letting the cells incubate in fresh growth media for 24-48 hours. This reduces the inflammation markers induced from differentiation. This may be similar for the LPS protocol. However, it seems like LPS is a confounding variable for your outcomes.
Interested to know if you find an alternative method.
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