Hi,
Does anyone know a method apart from doing FACS to isolate just the macrophages and not the others (neutrophils, B and T cells)? The simpler the better!:)
Thanks!

Adherence to plastic should work. Place the cells in 37°C tissue culture media (should contain at least 10% FBS) in a tissue culture flask in the incubator for about 2 hours. The monocyte/macrophages should stick to the plastic and the rest of the cells can be removed by gentle agitation of the flask to re-suspend the cells in the medium.

Adherence to plastic is the simplest procedure, and surprisingly effective for macs; density gradient centrifugation using Ficoll or Optiprep is good if you want it quicker (~15 mins as opposed to 2 h); enhanced density gradient using Ab-coated density beads for negative or positive selection takes longer (~35 mins), but gives very high purity, as does magnetic sorting using Miltenyi or similar MACS beads.

Also, note that adherence to plastic will give you both macs and neutrophils - if you get PECS from a naive (unelicited/unactivated) mouse, you can expect to get ~3-6x10e5 macs; a thioglycollate elicited mouse will have 10-times the number.
Using ice-cold sucrose for flushing the PECS has worked best for me.
All the best!

Hi, it's interesting that you are trying to isolate monocytes, while I am trying to isolate neutrophils, but from rat peripheral blood.

Does anyone know the protocol?
-----Neutrophils isolation from rat peripheral blood?

Hi all!!!
Does anyone know a protocol for isiolation of neutrophils from the peritoneum?
I extract the cells by injection of thioglycolate 2.9% into the peritoneum and after 4 hours draw the cells. By using this protocol I get a mixture of neutrophils and monocytes. I want to seperate between the two. How is that done?
George

If you want to accurately differentiate between monocytes and neutrophils in suspension, you can do it by FACS. Differential cell counts based on staining and nuclear morphology is another way.
For FACS, using 7/4 and Ly6G Abs is a good way to differentiate the two.

I have problems with white blood cells after I collect peritoneal macrophages. I am injecting 4% thioglycollate ip and collect cells from peritoneum after 4 days. I could not get rid of round cells I also saw one macrophage was eating. I am checking proinflammatory cytokine and it gives me false response I think. What do you recommend? You are using serum free for the first hour only them after removing the other cells so you use medium with serum?
thank you
good luck

Aged 4% thioglycollate in saline works very well. Ready made formulations tend to differ, but the simple recipe with Sigma thioglycollate has worked just fine. The 'elicited' macrophages differ from 'native' ones, but they do not have an 'activated' macrophage phenotype.
The aging part can be done by storing the autoclaved sterile thioglycollate in the refrigerator over several weeks (2-3 atleast), preferably several months. I'm not absolutely sure why the aged version is better, but theres years of anecdotal data from labs all over the world on this one!