I am currently looking at specific immune markers in the lungs (BAL cells) and whole blood of human subjects by flow cytometry. We are trying to determine the expression levels of different cell types present:
CD3+CD4+ or CD8+ = T cells
CD3- CD56+ = NK cells
CD14-CD16-CD83+ CD49d+HLADR+ = Dendritic cells (not distinguishing between different DC subtypes)
CD14+CD16+HLADR+CD49d+ =monocytes/macrophages
CD14+CD16+HLADR-CD49d- = Neutrophils
I have researched a bit and found that expression of CD49d and CD16 can distinguish between neutrophils and eosinophils. While we are not interested in eosinophils we want to ensure that we can distinguish them from neutrophils. However, while CD49d is definitely not expressed on neutrophils, there is no consensus on whether it is present on macrophages. In a number of articles there may be low or no expression of CD49d on Macs.
So my main question is:
Can anyone suggest better markers to distinguish between neutrophils and macrophages
I have read that neutrophil elastase may be a good marker but I also read that Macs may also produce elastase. What about CD66b? Does anyone have any experience with any of these markers in human samples?
We are limited by the number of colours we can run on our cytometer and the number of cells we receive so ideally we would want only one or two markers that can distinguish between the two cell types
Also for elastase – can anyone suggest a good company that supplies conjugated neutrophil elastase Ab (conjugated to any fluorochrome that falls in red or green laser channel)
Thanks very much!!
You can choose from the extensive list here
http://onlinelibrary.wiley.com/doi/10.1002/0471142735.ima04as80/abstract
CD14 and CD16 are ideal for monocytes (with the more mature monocytes expressing CD16 - see papers by Joan W. Berman) but may not be ideal for macrophages, as macrophages express low levels of both of these markers. Also, I have seen studies that find CD14 and CD16 on neutrophils so I am not sure those markers are ideal. Traditional markers for monocytes/macrophages are CD11b, CD14 and CD68 - with CD68 being most common in IHC and immunofluorescence as it requires cell permeabilization.
However, if you don't mind permeabilizing your cells, CD68 does work for flow cytometry, although you likely have to adjust your workflow. There are also some papers that suggest CD68 is present on neutrophils and some other cells, but it is highly expressed on macrophages and you should be able to distinguish them by the MFI in that case.
I am not familiar with CD66b, and have never seen it used as a macrophage marker for MDM or perivascular macrophages, which are mostly what I work with. Other markers that people use for macrophages are CD163, CD206 and CD71, the transferrin receptor, (which is expressed abundantly in macrophages an minimally in monocytes) - but I believe that CD71 is also present on neutrophils at higher levels.
You might also try a non-traditional marker, such as the chemokine receptor CCR5, which is highly expressed on macrophages but to a lesser degree (some studies say not at all) on neutrophils and only minimally on monocytes.
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