Hi,
I am wondering about the surface levels of CD66b on unstimulated human neutrophils, analyzed ex vivo in whole blood using flow cytometry. The literature appears controversial, ranging from a recent paper suggesting that CD66b is consitutively co-expressed with CD11b and CD16 on up to 99% of human neutrophils (independent from the activation status) to other studies showing that CD66b is only very weakly expressed in non-activated neutrophils. I am hoping to use CD66b,CD11b and CD16 to define human neutrophils in peripheral blood but would need to know if CD66b is really a suitable marker for this (any particluar clone that works best?).
Thanks for your feedback!
Christian
Dear Christian, the document attached bellow will be of great help to you when you carefully read it. Goodluck.
Hello Chritian.
A single surface marker may not be enough to determine a specific cell lineage. In particular, the cells show differential expression patterns to a marker. This is why there is no reliable marker for unactivated neutrophils so far. Due to either different aims of the studies or careless analyses, some hasty and bad science have been accumulated in the study of cell lineages in immunology and cancer stem cell biology.
You may be a right person to resolve the problems by comparing the staining patterns of known cell lineage markers in both a strict, active, unactivating condition of the cells (probably using metabolic inhibitor from the time of the cell recovery within a tube) and activated cell condition to clarify what people miss in many dubious articles.
Best wishes.
Directly isolated neutrophils express very high levels of CD66b. It is also expressed by eosinophils. See supplemental figure S4H in this recent Immunity paper
Article Developmental Analysis of Bone Marrow Neutrophils Reveals Po...
Dear Emmanuel, Sang and Ben,
Thanks so much for your feedback and the helpful information. I will go ahead with my own stainings soon and will post my findings if you are interested.
In the paper cited by Emmanuel, CD66b and CD11b are clearly described as neutrophil activation markers: "Both markers are stored in granules of the neutrophil and are expressed at the cell surface upon activation and degranulation." More recent papers, including the one cited by Ben, suggest constitutive expression of CD66b, even in resting neutrophils. I feel that both views might be correct since storage in granules can be seen as constitutive expression, however I would be interested in surface expressed protein which might be a feature of activation. Always interested in your thoughts, thanks again.
I have personally looked at freshly isolated neutrophils and eosinophils from whole blood and can confirm that they express abundant CD66b. I cannot rule out that our isolation protocols do not induce some form of spontaneous activation, but I do not favour that interpretation. Certainly, I would expect that you will also see good expression of CD66b in your own assays.
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