Unlike human, mouse blood is not a good source of monocytes due to its extreme low percentage. Usually people collect murine monocytes by sorting bone marrow with CD11b(+) CD115(+) F4/80(low). Based on Ly6c expression, murine monocytes can be further categorized into two subtypes. It is also reported that there is a splenic monocyte reservoir which contains 1-2 millions monocytes. However, bone marrow source can easily beat that number. Culturing monocytes with M-CSF for 4-7 days, you will have macrophages.

Thanks Xiao for the reply. We noticed it today that the monocytes were very few in mouse blood when we isolated. The M-CSF which is to be added for macrophage differentiation; is it specific for splenic or bone marrow monocytes or for monocytes from any source.

Monocytes from every source are very similar. Basically they are the precursors of tissue macrophages and dendritic cells especially in inflammation (in steady states, tissue mac and DC proliferate and keep the homeostasis by themselves). As long as the monocytes reach different tissues, they will differentiate to different macrophages directed by the microenvironment cue. So M-CSF provides a cue to direct the differentiation to M-CSF-induced macrophages.

ok i understand. We will try the experiment and keep u posted after achieving results.

Thanks for the help.

Is there an optimal concentration of M-CSF to use for particular amount of monocyte cells??

After differentiation into macrophages in 4-7 days, if we want to validate our results, can you suggest me some idea how to recognize or be sure that the differentiation went well and what we obtained are macrophages.

Based on Siamon Gordon's protocol, 0.5-1.5 ng/ml. Based on our experience, you can raise the concentration to 10 ng/ml.

There are a couple ways to identify macrophages. The two simpliest ways are: a good pathologist can tell them by morphology; FACS can tell a significant upregulation of F4/80. By the way, the cells will become more sticky to the culture dish. I suggest you to use Petri dish (not regular cell culture dish) so you can harvest the cells by treating them with 10 mM EDTA and 4 mg/mL Lidocaine-HCL for 10 min followed by quench with same volume of complete culture medium.

I get my macrophages by washing bone marrow out of the femur bone, then grind it up and RBC lysis like what you do with a spleen, and culture in RPMI with M-CSF. I also suggest that you change the media and wash away the floating cells every 2 days.

Thanks for the replies. @Yi Wen: my experiment is to isolate monocytes from peripheral blood and to differentiate them into macrophages which will be quite different than getting macrophages from bone marrow. As per Xiao suggestion we will add M-CSF and see what happens!!!!

You can try this. Inject mice with thioglycollate to elicit macrophages. 4 days after intraperitoneal injection of 2–4 mL of 4% sterile thioglycollate solution, inject 10 ml of PBS in peritoneal to collect macrophages by peritoneal lavage into ice-cold PBS. Resuspend the cells in appropriate media.

@Aravind: it seems that this discussion is a few months old now, I am very curious whether you were able to isolate these blood/splenic monocytes and differentiate them into functional macrophages? Did you perform FACS-analysis, NO-assay on them?

@Kristiaan: Yes we did isolate mouse monocytes by using magnetic separation from STEM CELL technologies but the recovery was only 45%....we exactly don't know how many days we need to differentiate and the morphology of macrophages was not that accurate even after 5-6 days. We don't obtain lot of blood from a single mouse and we need to pool blood form different mouse so we thought this experiment would take more time n money.....hence we dropped it and now working with a cell line. Once we obtain all the results, we will think of validating them in primary macrophages

Thank you for your answer! Maybe tis paper will assist you if you decide to turn back to your original idea. Regards http://www.ncbi.nlm.nih.gov/pubmed/19644120