Hi Mohammad, You followed the protocol and did nothing wrong. What you have found here is that the literature has oversimplified M1/M2 polarisation to the point of being almost completely wrong. So the answer to your question is yes, this is normal, though lab and donor dependent (also monocytes are extremely heterogeneous). If you are set on using human macrophages, then bone-marrow derived cells are recommended though more difficult to get and can be expensive to buy. Otherwise, I would say try reducing time in culture (as there will be lots of death at 8 days) to a max of 3-5 days, though note that you will never get a homogeneous population. I would recommend that you forget the word macrophage and remember the word monocyte for your experiments. Monocytes are not just precursors to macrophages, they are an effector cell in their own right, the macrophage differentiation (mainly defined by morphology) is usually a by-product of their activation (or in the case of parasite infections/ gut macrophage replenishment it is required). Just think this: when a monocyte is recruited to an inflamed tissue or tumour does it need to wait 8 days in order to work, or will it start working instantly and IL-4 or TNF signalling will alter this over time (bearing in mind that signalling events are 1-2h), this time will be unlikely to last 8 days considering the bone-marrow production rate in inflammation (the cells will likely be dead by this point, especially in vivo/in situ). Also ask whether morphology is more important than function, what readout do you want - this will be project dependent - but if you want to see inflammatory vs anti-inflammatory cytokine production, monocytes can do this within a few days of culture for sure. For me when writing grants, it is very easy to justify work on monocytes using these simple truths, the human macrophage field is a mess.

Hi Luke... Thank you so much for your answer