[Cytometry] about CD14-negative mature monocytes

mario picozza mariopicozza at hotmail.com
Wed Feb 26 04:39:40 EST 2014

Ok, let me
clarify and, I hope, add matter of debate.

Nuno is right, his combination of Abs is sufficient to identify
monocyte subsets. 

Also, I strongly suggest the reading of the consensus paper from the IUIS
committee (Ziegler-Heitbrock L., et al Nomenclature of monocytes and dendritic
cells in blood. Blood. 2010 Oct 21;116(16):e74-80) on the cytometric definition
of the subsets. The commitee states that non classical monocytes are CD14+
CD16++, where + and ++ denote a ten and a hundred fold expression above the
isotype respectively. Although this definition is not technically perfect, I
will discuss here what it roughly means, that there is a metric based on
isotype control (FMO looks the same) to assign monocytes to one or another
subset. For most individuals, and whatever clone you use, the degree of CD14 in
non classical monocytes partly overlap with that of negative cells. This is the
reason why one subset of myeloid dendritic cells (negative for lineage markers
including CD14-19-3 and 56) which expresses CD16  was early recognized
(Mac Donald, Blood, 2002) and why there is a debate about the nature (DC or
monocytes?) of so called slanDC. 

I think the solution straightforward, and, instead of making use of isotype controls,
solves the non classical subset separating it from the background (as recommended by Hulspas
et al., cytometry part b, 2009). Indeed, CD11c is usually used  to look at
myeloid dendritic cells. If used in conjunction to HLA-DR it can pre-select
myeloid mononuclear cells from both neutrophils and CD16+ lymphocytes. After
HLA-DR, it is a 2nd dimension to define all monocytes and myeloid DC together.
Now, gated on DR+ CD11c+, lets the CD16 vs CD14 plot speaks itself. 

As it can be intuited by the literature, there is a large amount of FCS data
bytes coarsely spread all over the world with a panel like "lineage (CD3 14 19
and 56), DR, CD11c and CD16", originally designed to select CD16 positive and
negative mDC (there are also a number of vendors selling so called lineage2).
As can be easily argued, this material can be re-analyzed by first gating on
CD11c+ DR+ mononuclear cells, than plotting in Lineage vs CD16 to visualize
mono-subsets along the CD14 vs CD16 rainbow (practically no lineage
positive cells other than monocytes express CD11c and DR together) and mDC (see
figure 1 A and B of my article, Picozza et al., Blood, 2013). 

P.S. Instead of CD11c one can use CD86 (in resting ex vivo conditions)

Mario Picozza, Ph.D.

Laboratory of Applied Dermatology

Istituto Dermopatico dell'Immacolata, IDI I.R.C.C.S.

via dei Monti di Creta 104, 00167

Rome, Italy

Date: Fri, 21
Feb 2014 22:06:03 +0000

From: cravista at gmail.com

To: TULUC at email.chop.edu; cytometry at lists.purdue.edu

Subject: Re: [Cytometry] about CD14-negative mature monocytes

Hi Nuno,


I saw your message on the Cytometry list. I wanted to ask if you know what

percentage of mature, circulating monocytes are CD14-negative and how can

one prove that those CD14-negative cells are indeed monocytes. If you know

of a paper that describes these cells please let me know.


Thank you,




>>>>>>>>>> Hi,



I will answer below.


As far as I know, the distribution of monocytes in the peripherical blood

is this:

- 80 % are "classical" monocytes with CD14+ CD16-;

- 10 % are "non-classical" monocytes with CD14- CD16+;

- 10 % are, let´s call "intermediate" monocytes with CD14+ CD16+
>>> this

population can be divided in two subpopulations, depending on the

expression of HLA-DR: one has high expression and the other one not so high;


All (or almost all) of the monocytes are IREM2 positive (CD300e) and CD64

positive as well. I think that all of them are CD36 positive as well...

Dendritic CD16+ cells also express IREM2.


So, if you want to study PB monocytes and you just have 4 or 5 colours,

just put CD14, CD16, HLA-DR and CD45.


I hope it helps.





Nuno Oliveira, IPOCFG - Flow Cytometry Division


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