How to differ macrophage from the other in tissue?

Hi all!,I want to know the amount of macrophage cells in inflammation tissue in mice skin, but I think that is not easy because it's difficult to differ macrophage from other WBC like neutrophil, and lymphocyte with custom dye. Anyone can give me suggestion to do it?
Can I use immunohistochemistry to do it? If it possible, what kind marker (antibody)I must use? Any other manner beside immunohistochemistry to count number of macrophage??

QUOTE (fiat29 @ Jul 7 2008, 09:00 AM)

Hi all!,I want to know the amount of macrophage cells in inflammation tissue in mice skin, but I think that is not easy because it's difficult to differ macrophage from other WBC like neutrophil, and lymphocyte with custom dye. Anyone can give me suggestion to do it?
Can I use immunohistochemistry to do it? If it possible, what kind marker (antibody)I must use? Any other manner beside immunohistochemistry to count number of macrophage??

Hi Fiat,

That's actually a pretty big question because whether or not a WBC is a 'macrophage' isn't really a binary thing. What I mean is that it is not a simple macrophage or not situation, but you can do your best to boil it down to one.

The problem is mostly in the transition from monocyte, or immature cell, to macrophage. However, if you determine that you only want mature macrophages the problem is much simplified.

First off, monocytes are suspension cells, whereas mature macrophages are adhesion cells.

Secondly, there is more than one marker that you can use to label macrophages. One in itself, in my opinion, isn't enough, but if you use more than one you're gold.

CD14 is a commonly recommended marker for macrophages, but I prefer Fc gamma receptors (CD16, CD32, CD64). While you will find FC gamma receptors on many different types of white blood cells, they have a much higher expression level on macrophages. Together, I think these work well for macrophage selection. Another marker would be CD116 (Granulocyte Macrophage - Colony Stimulating Factor Receptor) which has high expression on mature macrophages, but a low expression level on immature macrophages.

So, what I'd recommend is something like...

1: Use para-magnetic beads labeled with anti-FC gamma receptors to first harvest cells from tissue.

- This must be done before the tissue is digested, the reason being, that macrophages are typically NOT found within the tissue. Consider, Alveolar macrophages (macrophages found in the Alveoli of the lungs) they exist on the surfactant (lung fluid) found on the epithelial lining within the macrophages.

I have harvested them on occasion and it is not difficult. But you SHOULD harvest them before you digest the tissues. They lose adhesion to the tissue in the pressence of EDTA (1 to 5 mM in PBS).

Ergo, if you harvest cells that express FC gamma receptors, PRIOR to tissue digestion, there is a high probability that those cells are macrophages. This is not absolute but it is a VERY good start.

2A: (for quantification) Fluorescently label cells for CD14, and then use flow cytometry to count.

2B: (for harvesting and culturing) use para-magnetic beads labeled with anti-CD14 to seperate and collect macrophages.

2C: (Quantification and harvesting) Fluorescently label cells for CD14, and then perform a facs sort to collect cells that have a high expression of CD14.

If you want to be more certain about whether or not the cell is actually a macrophage, add a fluorescent label for GM-CSFR and then collect only cells that have a high expression of both CD14 AND CD116 (GM-CSFR)

I hope this helps. If you have more questions feel free to contact me through email. I'd be interested in hearing more about your research.

Good Luck.

Tim

How to differ macrophage from the other in tissue? - (Jul/07/2008 )