I have been using a CD34-PEcy7 reagent from Becton Dickinson (clone 581, class III epitope) to stain PBMCs for flow cytometric analysis. CD34 haematopoietic stem cells are not the only cells to appear 'positive' - monocytes are too (they all consistently have a delta GMFI above 100 units - I dont have the actual figure to hand). the samples were stained in the presence of human serum and 10ug/ml human IgG, using an FMO strategy except that isotype control antibodies were used in place of specific antibodies for the relevant flourochromes and acquired on a QC'd Aria. From the literature there is no evidence that monocytes express CD34 antigen and there have been a couple of HSC centred papers I have seen that monocyte staining is a false positive. This was revealed by comparing two antibodies from different vendors of the same clone - but they never revealed which vendors these were.
When looking at the data sheet for this antibody and many others in the market place they are almost all class III epitope antibodies and they only show representative staining derived from a lymphocyte (where there is a hint of some events with a higher FSC appearing positive) or cell line gate. Only one company, Miltenyi Biotec, does show gating derived from total PBMCs and here monocytes appear negative but this is with a different clone to the one above.
My understanding is that CD34 is a Ligand for L-selectin so is it unreasonable to expect some positive staining?
I plan to isolate CD14++ cells using MACS technology and plan to examine CD34 mRNA anyway but is anyone else working in this area that could shed light on this?
thanks,
Adam
Actually you have to measure it and find it by yourself whether it expresses two cytokines or not?
They shouldn't express CD34...however, I found in the past a small inexpecific signal after having my cells in culture and it was due a bad anti-CD34 antibody.
it is discribed that human CD14+ monocytes bind PE-Cy5 and PE-Cy7, so they could be false positive. i would change the colour combination and repeat the staining.
According to general understanding, No.
CD14 stains differentiated cells: Monocytes/Macrophages.
CD34 is found on progenitor cells.
But as always in science, you may see someting different because nobody has given it a thorough look. Not sure if you really want to go deeper into this, but here some suggestions anyway:
I understand, the staining is rather weak, compared to the majority stained cells?very weak staining could always be some sort of nonspecific binding (despite all blocking). Do you have another marker that should be completely negative (CD3 might be a candidate) Do REALLY thorough controls to make sure you are not chasing artifacts. Do isotype controls, switch colors, as suggested by Bastian. Sort some of these cells, do cytospin and have a look on morphology, to see if they differ from regular CD14 positive cells.
definivement yes, but at very low level.
20 years ago we developed antibodies against DC generated in vitro from monocytes cultured in GM + IL4.
Among more than 1,000 antibodies generated, we obtained 5 clones that have proven to be anti CD34. These antibodies showed that CD34 is always present on the CD14 + strong positive, and it increases with the DC maturation.
It's the same with the contaminating subpopulations mDC, pDC and basophil that are associated with the purification of monocytes.
Another CD is also very present and evolves in the same way; CD33
Really it CD14++ monoctes is lack of CD34 expression. In myeloid leukemias with monocytic differentiation represent a heterogeneous group of bone marrow disorders. Acute monocytic leukemias frequently lack expression of CD34 , which is more often seen in other AMLs, but commonly express HLA-DR, CD33, and CD64 with variable positivity for CD14.
Hy Adam, is correct to mention the rest of people about these cells (Classic Monocytes, CD14 ++) that theoretically shouldn´t CD34. On the other hand is very important both, clean the instrument (Flow Cytometer), to avoid any carry-over possibillities, as well as cleaning solutions that work (filtering), all this in the sense of avoiding inespecific reactions. Finally, this mesures at the mRNA level, I consider that it is the right thing way that will certify the expression or not of this molecule,
Good Luck.
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