Dear Samanta,

the best approach for definition of mouse blood monocytes is to use

anti-CD115 as a monocyte marker and

Ly6C and CD43 to define the subsets.

(Sunderkotter, J Immunol, 172:4410, 2004).

Do not use GR-1 because it also stains granulocytes. Also, both CCR2 and CX3CR1 are of limited use since the expression levels on mouse monocyte subsets show only moderate differences (Ingersoll, Blood, 115:e10, 2010).

Please note that the new nomenclature proposal defines classical Ly6C++CD43+ monocytes and non-classical Ly6C+CD43++ monocytes (Ziegler-Heitbrock, Blood, 116:e74, 2010). In addition, with these markers you can see an intermediate population (Sunderkotter as above).

Best of luck with the experiments

Loems

You can stain for CD11c using flowcytometry, which can distinguish between inflammatory and non-inflammatory monocytes.

Also do use use purified monocytes or PBMCs. If you use PBMC, the probability to get lot of background is evident.

Hi Samanta, i would suggest you to stain your monocytes with CD11b and Ly6C. The strategy is like following-

1. CD11b+ Ly6ChI (CCR2+ CX3CR1–)- classical or inflammatory

2. CD11b+ Ly6Clo (CCR2– CX3CR1+)- non classical/patroling

Hope this will help you. But if possible always measure ROS/NO within thses subsets to conclude about their nature. You can read this paper-

"The diverse roles of monocytes in inflammation caused by protozoan parasitic diseases"

Meru Sheel and Christian R. Engwerda, published in Trends in Parasitology, 2013

start with lineage negativ (blot 1), than show CX3CR1 vs CD11b and gate for double positive (blot 2; do you see CX3CR1 high and low monocytes with FACS?) and than show CCR2 vs Gr1 (blot 3).

Hi Paul and Debanjan, first of all I wanted to thank you for your help, I really appreciate it!

I will consider marking my cells with CD11c too, I hadn't thought of it before because I was so focused on the other markers, but it's a good idea.

I think I will start by trying the strategies proposed by Debanjan so that I can make the most of the data I already have and then I will try to improve the analysis with CD11c.

Thanks again!

When analyzing mouse blood monocytes by flow, I typically use the following gating strategy:

F4/80 (or CD115; CD11b would work too) vs SSC –> gate on F4/80+SSClow

Gating on the F4/80+ cells, I then look at F4/80 vs Ly6C (I prefer Ly6C to Gr-1, which stains both Ly6C and Ly6G) and can see distinct Ly6C-hi and Ly6C-low populations. If you follow this strategy, you could then gate on each population and show histograms of CCR2 and CX3CR1, or a dot plot of CCR2 vs CX3CR1.

Alternatively, if I gate on F4/80+SSClow cells and then look at CCR2 vs CX3CR1, I can distinguish and populations quite nicely, although this strategy doesn't utilize Ly6C.

I have just seen your answer, Alexander, thank you to you too. In blot 2 I see many CX3CR1 negative monocytes but not many CX3CR1 positive or double positive monocytes. CCR2 vs Gr1 blot is derived from double-positive cells of blot 2?

Not that simple, unfortunately. There are also myeloid progenitor cells, specifically MDSCs to be considered in your primary Dx. If focused on normal animals this is not a major problem, but if there is an inflammatory or neoplastic process on-going this can become challenging. In mice undergoing these processes most if not all of the "monocytes" are Gr1+ and arguably better defined as MDSCs. If focused on monocytes my first step would be to gate out garbage and apoptotic cells using a SSxCD45 plot. Then make a FSxSS plot and draw a monocyte gate. Of course this will include activated T-cells, such that your linage markers become useful. Note, that blockade of non-specific binding and choice of fluorochrome are critical when focused on myeloid cells. Thus, streptavidin and APC are not your friends. I am assuming that the linage markers are CD3, B220 and a strain specific NK marker. As such gate out the linage+ cells leaving you with monocytes, MDSCs and hematopoietic progenitors. One would then make a CD11bxGr1 plate which will provide up to 5 populations. We normally focus on Gr1brCD11b+ and consider these cells granulocytic MDSCs. The Gr1dullCD11b+ cells are considered monocytic MDSCs (Ly6cBr) and the Gr1-CD11b+ cells are considered potential monocytes and if if Ly6c bright defined as monocytes. These cells could then be subset on the basis of CCR2 and Cx3Cr1. It is assumed that CCR2 is used to identify inflammatory and CX3CR1 to identify resident monocytes. F4/80 and CD68 are also frequently incorporated; however, we do not find them especially useful. In general we consider a cell that is CD11b+Gr1negativeLy6cBr to be a monocyte. This last statement has potential to engender considerable controversy. Looking forward to all comments.

Dear Samanta,

the best approach for definition of mouse blood monocytes is to use

anti-CD115 as a monocyte marker and

Ly6C and CD43 to define the subsets.

(Sunderkotter, J Immunol, 172:4410, 2004).

Do not use GR-1 because it also stains granulocytes. Also, both CCR2 and CX3CR1 are of limited use since the expression levels on mouse monocyte subsets show only moderate differences (Ingersoll, Blood, 115:e10, 2010).

Please note that the new nomenclature proposal defines classical Ly6C++CD43+ monocytes and non-classical Ly6C+CD43++ monocytes (Ziegler-Heitbrock, Blood, 116:e74, 2010). In addition, with these markers you can see an intermediate population (Sunderkotter as above).

Best of luck with the experiments

Loems

hey samanta, we are talking about a staining in mice, right? to be honest, i never saw a convincing CX3CR1 antibody staining (and i tried a couple of antibodies, which were reported in other publications to work). so, i would be careful with the interpretation concerning your CX3CR1 staining.

the best staining for monocytes is still: CD11b vs CD115 and take double positiv cells; than you can go for Ly6C (as matthew stated (but Gr1 is also ok, you don't have to buy a new antibody)) vs CD11c (as paul stated) or CCR2. this should be ok. but i guess CD115 is indispensable, at least if you want to perform this staining in your lab routinely. do you use the CCR2 antibody from R&D 475301? do you get a nice staining?

but if you want to save some of your existing data, than i would gate for CD11b+ cells, show them in FSC vs SSC (as meredith stated) and gate out all SSC high cells. take these SSC low cells and have a look for Gr1 vs CCR2. it's not clean, but maybe the best if you cannot rely on your CX3CR1 antibody

Easiest would be use CD115 or CD204 to identify total monocytes, then use Ly-6C to identify subsets. never use CD11b alone to identify total monocytes, it is not specific. also do not trust SSC versus FSC as it is hard (also depends on the flow cytometer you use) to clearly separate monocyets from others, particularly nonclassical are smaller and could come together with lymphocytes and more importantly they could change scatter pattern in some mice such as apoE mice on HFD (see Circulation 2009 May 26;119(20):2708-17). Good luck!

First, I wanted to thank all of you that have put your efforts to help me. I want to answer all of your messages, so here we go:

Answer to Alexander Mildner: Yes, it’s staining in mice blood. I think the same about CX3CR1, it doesn’t seem to stain well, I don’t know why…

I tried to use CD115 before, but since the cytometer is not in our lab and I have to move to another part of the city, it seems to lose some signal due to the delay until I process and analyze the samples. That’s the reason why I decided to replace CD115 and use CD11b, because for what I learnt from literature, it seems to be more stable than CD115 if you can’t get access to the cytometer immediately.

Yes, that’s the CCR2 antibody that I use. I’m pretty happy with the staining I get, it seems to work quite well, unlike CX3CR1…

I think it is a good idea to establish the gates as you have advised me. I will surely try it.

Answer to Meredith: Hi Meredith and thanks for this thorough explanation on how to analyze blood monocytes. It will help me a lot!!! I’ve been having trouble understanding the difference between GR-1 and Ly6C, but you have also explained the differences between the two (sometimes finding clear information is difficult even reading many papers…), so thanks for that too :).

Answer to Matthew: Hi Matthew, thanks for your help. I’m using a flow cytometry antibody. As I told Alexander I agree with both of you that CX3CR1 antibody doesn’t work well… At the beginning I thought that I had processed something wrong, but when I saw that the other antibodies worked well I started to suspect that there is some type of issue with this antibody, still not sure what…

Thanks for the recommendation of using a neutrophil antibody. The decision of not including more antibodies came from the fact that the wavelengths of the antibody fluorophores would overlap, so I tried to minimize the number of antibodies. Usually it is hard to choose which antibodies will be the best…:(

Answer to James: Hi James, and first of all, thank you for your help. We used healthy animals but also SOD1G93A transgenic mice, which is the most used mice model for Amyotrophic Lateral Sclerosis. The causes of this disease are unknown, but it is know that there are some inflammatory and of course degenerative processes happening in the nervous system and the muscles, so, yes this can be a problem, but as what I really wanted to know was the approximate % of inflammatory and non-inflamatory monocytes it is ok. The lineage markers include: CD3, CD45, and TER-119, we would need one more for NK cells then. I think the protocol you recommend is very interesting too, but surprisingly in some of the papers I’ve read monocytes are considered Lineage positive instead of lineage negative which leads me to confusion, because they appear to be in the limit between progenitor and mature cells. I think this is because they have used a cocktail which includes some monocyte markers.

Answer to Loems: Hi Loems and thank you for your advice. Now I’m pretty sure that GR-1 won’t help me much, as many of you have told me. I wasn’t aware that CD43 was so important, so thank you, I will check the literature again. Some of the papers I read surprised me very much because apparently they could “easily” distinguish both monocyte subsets using CCR2 and CX3CR1 but it seems that clearly it’s not like that. Thanks again!

Answer to Huaizhu: Hi Huaizhu, and thank you for your answer. At first, I considered using CD115, but as I said before, it seemed to lose signal if I didn’t get to the cytometer fast, which I couldn’t do because it is not located in our center. Now I understand that CD11b is not that reliable seeing all your comments, and yes, I have seen that the cytometer we use isn’t able to clearly separate the two subsets. I was very disappointed because in some papers it seemed easy to do, but as you said, it depends on the cytometer you have. Thanks for the help and the paper you have recommended!

Ditto the CX3CR1 antibody issue. We've had to resort to using CX3CR1-gfp reporter mice to look at expression by flow...probably not the most practical thing. Good luck with the staining!

Also keep in mind that Ly-6C is not specific on monocytes either, granulocytes also express Ly-6C. Therefore, you need to use a monocyte specific marker (or multiple markers) to define total monocyte first. It would be "okay" to use Gr-1 (Ly-6C would be better) to define monocyte subsets (again on the top of monocyte marker). The differnece between Gr-1 and Ly-6C is that Gr-1 reacts with both Ly-6C and Ly-6G. While Ly-6G is expressed on granulocytes specifically, Ly-6C is expressed on monocyte subset, granulocytes and some others.

Something seems off with the linage markers. CD3 as T-cell is good. CD45 would be all hematologic cells and not useful. I suspect you mean CD45R or B220 for B cells. If you are using CD45 you have no marker for B cells. TER-119 stains erythrocytes and is useful if you are having problems with lysing rbcs. Using CD45 (not CD45R) should obviate the need for TER-119. Of course its nice to know if the garbage is leukocyte fragment or rbc fragments so its a bit useful.

Please look at our previous publication in Journal of Immunology 2011, by Kurotaki D, Uede T, et al. In our introduction and discussion section, you may find your answer.

Thanks for the explanation about the differences between GR-1 and Ly6C, Huaizhu. As I said before, I was a little confused about the differences as I'm not a immunologist. I will take it into account from now on.

That's right, I was referring to CD45R or B220, my mistake, sorry. In relation to TER-119, when we started the experiment we had some issues with the erythrocyte lysis, so we thought it will help us solving that. Thanks again James.

Thank you for recommending your publication, Toshimitsu. I'm going to read it right now.

Hi Samanta - I can see the science is being answered above, so I'll answer the bit about Kaluza. I train staff & students in Kaluza at a daily basis here at the College of Medicine at Swansea University. Once you have your data files I would be happy to help you set up a template so that you can drag and drop files from new experiments with all the gates already set up. The best way to do this would probably be to share a dropbox & skype. Send me a message here on Researchgate if you want me to help you set up a template. Best regards, Ina Laura

Gate for CD115 (this will also take out neutrophils), then Ly-6C and CX3CR1. Since there is no antibody to mouse CX3CR1 that works, you need to use the GFP knockin mouse (see refs below). Look at C.C. Hedrick's and Frederic Geissmann's papers for correct gating. CCR2 is a great marker to see the classical monocytes.

1. Jung, S., J. Aliberti, P. Graemmel, M. J. Sunshine, G. W. Kreutzberg, A. Sher, and D. R. Littman. 2000. Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Molecular & Cellular Biology 20: 4106-4114.

2. Geissmann, F., S. Jung, and D. R. Littman. 2003. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 19: 71-82.

We routinely classify mouse monocytes in mouse blood: We define monocytes as CD19negative CD11b positive Ly6G negative NKp46/NK1.1 negative cells.

We classify them into six groups:

(I put them in the order of expected frequencies under baseline conditions):

CD11c-, Ly6C-

CD11c- Ly6Cintermed

CD11c+ Ly6C-

CD11c- Ly6C high

CD11c+Ly6C interned

(very little, if any: CD11c+ Ly6C high)

From my perspective you would need the following markers: anti-NK marker (for B6 you might take NKp46 AND NK1.1. on the same channel), Ly6G, CD11b, CD11c, Ly6C, and best an anti-CD45 as 'threshold channel' .

Contact me for further details.

Thure

What if I do:

CD45+/SSC LOW (exclusion of neutrophils, high SSC)

CD115+/ CD11b high+ / ( Gr-1 ) also said LY6C/G+

Do I still need to exclude lymphocytes or NK??

they should be CD11b negative, right?

Thanks in advance

We start by gating on CD115+. There is no antibody for CX3CR1, so you cannot use this marker. Ly-6C is useful (high and low, see Ziegler-Heitbrock). if you have a good CCR2 antibody like the one from Matthias Mack, this is an excellent marker for the Ly-6C-hi. there is no positive marker for the Ly-6C-lo subset. Monocyte subsets change when the mice are on an atherosclerosis background. CD11c+ monocytes appear, and some express CD36, see Ballantyne's work.

Thank you all for your help. Finally with all your answers I think I got satisfactory results for my analysis :)