Many thanks to all who replied to my request! And there were many!! You
have greatly simplied our search for a different marker(s) to distinguish
monocytes from neutrophils.
For those who have or will have a similar problem, I have listed the replies
below for all to see.
Julie Oughton
Oregon State University
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Try CD64, it should mark monocytes. This is probably something you will
need to markers to really identify the monos. CD13 is also a good marker,
I would still use a CD14.
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CD16, CD36, CD64 are the common monocyte markers, Some of these CD's are
shared by granulocytes, except CD36 which is shared by platelets ( no
problem in open gating) and fetal RBC's.
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Our studies in the last year have convinced me that CD163 is the best single
marker for human monocytes, followed by CD64, then CD14. The combination of
CD163PE and CD14FITC or CD14PE maybe superior to single reagents if your
desire is to identify the most monocytes. The first two markers can be
obtained as PE and FITC conjugates from Maine Biotechnology Services
(www.mainebiotechnology.com <http://www.mainebiotechnology.com>) and CD14
can be obtained from any of the usual suspects.
CD16 would be my choice for defining neutrophils (eosinophils are CD16
negative).
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CD14 is a great marker for human monos. If you use the celltracker on your
XL, you'll see the 14 positive cells go right back to the mono population on
scatter. You might get better resolution of the monos on a 45/SS plot
instead of FS/SS.
The Coulter CD14 clones are all very good. Have Super Dave get you a sample
of the IOTest RM052 clone, or the Coulter 322-A. Either are very good
reagents.
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I would definitely recommend using the CD14 in lieu
of CD11b. When I was doing lymphoma studies on adult peripheral blood,
there was almost always a small population of lymphs that were distinctly
CD11b positive. When looking at human blood, there is a pretty typical
distribution of cells when looking at CD45 vs. SSC. She could set a gate
with this plot that would exclude granulocytes, non-leukocytes, and debris,
and then look at the gated population (lymphs and monos) for CD14 positive
cells. The lymphs should clearly be negative.
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CD11b will be positive on some lymphocytes depending on the activation
status of the lymphs (mostly NK's which could have similiar properties in
FSC/SSC as the monos). Can I make a suggestion? How about CD45, SSC (med),
CD14, CD64? The monos can be well well worked out this way
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I use CD14 for years now. To my opinion it may be the best antigen for
monocyte targeting as it has a rather high expression. My gating stragety
also includes the scatter characterisics because this helps to find the
right population.
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Fresh human leucocytes segregate very well on linear forward v side-scatter.
With that, use CD14 for monocytes and CD16 (or better - CD16b - less labile,
more specific) for neutrophils - but don't expect neutrophils to survive
freezing.
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We use Leucogate (CD45/CD14) to discreminate Ly, Mo and Gr.
see for example <http://www.diatec.com/product.asp?pg_id=45>
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In my opinion CD14 is a really good marker for monocytes, check out the BD
monoclonal AB ( catalog # s- 347490, 347493, 347497, 340585, 340436) The
flourochromes attached are Pure {no flourochrome-in case you want to label
with a secondary Ab} ,FITC,PE, PerCp and APC respectively. You can also
find corresponding Isotype ctrls on the site (IgG2b).
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