Macrophage collection from mice

5 posts / 0 new
Last post
abcd
abcd's picture
Macrophage collection from mice

Hi,

I want to use murine macrophage cells for phagocytosis assay. I want to know what is the difference between peritoneal macrophage and bone marrow derived macrophage for this type of assay. I have seen both types of cells being used in literature but is there any specific nature of any one type of macrophage than the other? I would really appreciate if anyone in the filed can give me some details.

Thanks.

marcus muench
marcus muench's picture
The two macrophage

The two macrophage populations are different in their 'state of differentiation' or 'activation'. There are some resident macrophages in the bone marrow, but most of the cells you may isolate from the marrow would be called monocytes, they are much smaller with a lower ratio of cytoplasm to nucleus. Peritoenal macrophages are very large cells with many cytoplasmic vacuoles. I beleive there are significant amounts of M-CSF produced in the peritoneum which, in part, leads to the further differentiation of monocytes into peritoneal macrophages. If you grow bone marrow cells in M-CSF you will in fact produce a large number of macrophage cells in culture. Howeever, anytime you culture something you must be aware that you will likely affect gene expression and thus function. This is even true for freshly isolated cells cultured for even less than a day. Whether this matters depends on your specific question.

There are antigenic differences between marrow and peritoneal cells, consistent with the notion that these cells have unique gene and protein expression profiles. In other words, they are likely ot have functional differences. The F4/80 antigen (http://www.bdbiosciences.com/ptProduct.jsp?backLink=ptProductList.jsp&backName=Product%20List&prodId=429054) for instance is highly expressed on peritoneal macrophages.

Sorry, I haven't performed the phagocytosis assay, but I would guess the peritoneal fraction of cells is easiest to isolate in relatively pure form and purity of differentiation state (your going to get lots of different cells from the marrow). Isolation by adherence to plastic will likely give you predominantly macrophages as the peritoneal B cells etc will be easily washed free.

Good luck.

Soudabeh
Soudabeh's picture
abcd wrote:Hi,

abcd wrote:

Hi,

I want to use murine macrophage cells for phagocytosis assay. I want to know what is the difference between peritoneal macrophage and bone marrow derived macrophage for this type of assay. I have seen both types of cells being used in literature but is there any specific nature of any one type of macrophage than the other? I would really appreciate if anyone in the filed can give me some details.

Thanks.

The peritoneal macrophages are very effective and easy to produce and isolate. Inject 1ml of aged thioglycolate (from Sigma ) into the peritoneal cavity 3 days before collection. Collect macrophages from the peritoneal cavity using pasteur pipet and warm media. You might have some contamination with neutrophils.

Guy Sovak
Guy Sovak's picture
I tryed the assay that

I tryed the assay that Soudabeh mentioned some yers ago, it works prefectly.
You can try to stain for a Non Spesific esterase with counter staining of Eosin.

samm
samm's picture
Soudabeh wrote:abcd wrote:Hi,

Soudabeh wrote:

abcd wrote:
Hi,

I want to use murine macrophage cells for phagocytosis assay. I want to know what is the difference between peritoneal macrophage and bone marrow derived macrophage for this type of assay. I have seen both types of cells being used in literature but is there any specific nature of any one type of macrophage than the other? I would really appreciate if anyone in the filed can give me some details.

Thanks.

The peritoneal macrophages are very effective and easy to produce and isolate. Inject 1ml of aged thioglycolate (from Sigma ) into the peritoneal cavity 3 days before collection. Collect macrophages from the peritoneal cavity using pasteur pipet and warm media. You might have some contamination with neutrophils.

Using ice cold sucrose solution (instead of PBS - see other posts wrt macrophage isolation) considerably reduces any neutrophil contamination. Neutrophils peak with different kinetics than macrophages on thioglycolate elicitation.