Hi Valerie

In my experience you have part of your cells differentiated into macrophages. With M-CSF they have this look of sunny side-up fried eggs. But you have many undifferentiated cells also, and some more activated macrophages (the ones that are more elongated). It's normal that not all monocytes differentiate into typical macrophages. Maybe wash them with PBS a couple times to remove the non-attached cells.

Hi Bruna,

Thanks for your answer! I think washing them is not preferable in our experimental set-up, but I will discuss it with my supervisor. Do you also know by chance how I could improve the differentiation? To get more differentiated monocytes? Or is that dependent on the quality of the monocytes?

It varies a lot with the donor. But they are very sensitive cells, don't like old RPMI for example (we once had problems differentiating macrophages because of a change in the RPMI lot). If you mix 5% FBS and 5% human AB serum, it usually helps.

Naive monocytes are very sensitive to changes in their environment and may even differentiate during cell passaging due to a slightly hard blowing of the pipette. The cells morphologically exhibit protruding pseudopods and a somewhat enhanced ability to adhere to the wall.

Okay, thanks for your answers Kaifang Wang and Bruna Cunha de Alencar !

Hi Valerie,

IL-6 is a key cytokine in the differentiation of monocytes. Try to add increasing amounts of IL-6 starting with 10 pg/ml to your cultures and see what happens.

I think you could check whether they have been differentiated and polarized into M0, M1 or M2-like macrophages or not, using flow cytometry. You can use specific markers like CD86, CD40, CD163 and CD206 to observe your results.