I am trying to culture macrophages from the bone marrow of mice. I always worked with macrophages and had no problem culturing them but recently it has been hard to get them as most of them do not attach to the plate and die after one day (They look small). After isolating bone marrow cells and incubating them for 1 day, I collect non-adherent cells and incubate them for two more days to get macrophages. Here is the image of my cells (40 x) that I am expecting to see macrophages. (The black dots are the background of the microscope).
Hi Sahar :)
a few questions for better understanding:
- Do you use freshly isolated BMCs or did you thaw frozen ones?
- Whats your concentration of M-CSF or L929 S.N.?
Additionally, you mention that you culture the BMCs for 1 day and afterwards collect the non-adherent cells to culture them for additional two days. Is there a specific reason for this? Most protocols you see (and what we do as well) is culturing BMCs for 7 days in presence of rM-CSF/L929 S.N. with medium exchange in-between (day 3-4), keeping the adherent cells. From my experience, BMCs/BMMs will only start to adhere roughly at day 2-3 of culture. Therefore, it might be the case that you simply remove most of your cells "too early".
See for example:
https://pubmed.ncbi.nlm.nih.gov/23851980/
Hope this already helps :)
Best regards
Felix
Thanks again Felix.
Probably the reason is to remove all other bone marrow cells after one day and keep only monocyte and macrophages. I always cultured them like this protocol and could see very confluent and beautiful macrophages but recently I don't know what is happening in my cells or media.