Dear Gabriela,

CD206 is for sure a suitable marker to detect M2 macrophages in the tumor setting. You can also use CD163 (M2 Mac in tumor), CD80 (M1 Mac) or CD86, as you know marker of APCs. In the link below you can find a nice paper showing the phenotype of neutrophiles and the monocyte/macrophage lineage in the tumor microenvironment.

Hoping that everything will be fine,

Best

Damiano

http://www.ncbi.nlm.nih.gov/pubmed/23065796

Gabriela- Why not measure MPO aciivity in the spheres?  Those with neutrophil attachment should show measurable MPO activity.  The MPO analyses are used in studies of inflammatory bowel disease in the colons of mice and when there is neutrophil infiltration of the bowel we see big increases in the specific activity of this marker.

Hi! Thanks a lot Florian and Damiano for the valuable information, I will follow your recommendations as well for M1 and M2 macrophages markers that is also important for me.

Hi John, MPO assay seems interesting to confirm neutrophils infiltration. Do you use a kit? or how do you measure MPO activity? is this a method of high sensitivity? because if I use this assay I would prefer measure MPO activity per sphere (500µm diameter aprox), do you think it would be enough? Thank you very much.

Hi. You can try with a CD16 marker wich is specific of neutrophils, macrophages, NK, mast cells.

I attached you a table with Human CD antigens.

Hope this helps.

Siince the tissue you're dealing with are tumor spheres, infiltrated monocytes are more likely to have differentiated into macrophages (either M1 or M2)...so the best markers will be CD68 for both macrophages phentotypes and then you might want to add also iNOS for M1 and CD163 or CD206 for M2. To stain neutrophils, instead, you can use CD15 together with CD16 (double positive staining)...but if you use CD16 be careful because is also expressed on macrophages, so neutrophils would be CD15highCD16highCD68- while macrophages would be CD68highCD16+/low

there isn't a kit for MPO activity.  Just PubMed "DSS colitis AND MPO" and most papers will have the details.  It should be possible to measure activity/sphere knowing the number of spheres added.

Monocytes will be CD163 positive, as will be macrophages with higher expression, along with CD206.  PMNs normally CD15 would work, but as you point out many CD15 antibodies to react with some carcinoma cells.  CD64 would be on both, but lower expression on PMNs compared to monocytes.  As above the addition of CD16 might help, but it is also expressed on some NK cells and monocytes and is shed with PMN activation.  MPO as above can be useful, as a few years ago we assembled a kit for quantitation of hENT1 for AML blasts and successfully used MPO as a gating reagent.  CD14 I find occasionally confusing as it is not present on all monocyte subsets and can be expressed at low to moderate levels on neutrophils.  Thus I would use a combination of CD15, CD33, CD64, CD163, CD206 and MPO.  Feel free to contact us at [email protected].

Hi! thank you all for your help, I really appreciate it. :)

Hi Gabriela

for monocytes/macrophages visualised by immunohistochemistry I have used MAC387 antibody in the past for other studies (there is a suggestion it may stain some neutrophils also, you would need to check). What species are you working in? 

Hi Sean, I am working with monocytes and neutrophils isolated from human buffy coat :)

OK Gabriela

I only have experience in tissue (histopathology) samples sorry

Hi Gabriela,

For human neutrophils, combination of CD14, CD15, and CD16. Although people tend to use CD11b and CD33, it can be expressed by other cells such as NK cells.

You should be aware that MDSCs can express these markers as well where the main difference between them and neutrophils is immune suppression.

You can read the following review so it can give you some idea

Pillay et al Cell Mol Life Sci (2013) 70:3813-3827

http://download.springer.com/static/pdf/820/art%253A10.1007%252Fs00018-013-1286-4.pdf?auth66=1406478572_b4cd8b63f0e8d39ff6afa94b4102c6f2&ext=.pdf

The CD68 antigen is highly glycosylated. Take care when using CD68 as a marker for neutrophils as the different anti-CD68s may vary in their specificity.  PGM1 is a good antibody that does not appear to recognise monocytes.

I guess Gr-1 and F4/80 are pretty specific granulocytic and macrophage markers you should try. Goodluck

For macrophages, specific markers you should try for are CD11b and CD18. Neutrophil markers include CD15 and CD16.

In addition to CD14 and CD 15, the CD-68 is widely regarded as a selective marker for human monocytes and macrophages and is commonly used in human pathology studies. In neutrophil I think  CD 45, CD11 c and PECAM (CD31) are  more useful than MCF7

In our case we use GR-1 to label murine monocytes and neutrophiles, ae also use f4/80 to label macrophages!! If you work with paraffin sections, in the case of F4/80 you should make an antigen retrivel with enzymatic reaction (pronase) instead of pressure cooker. 

Try galectin-3 (MAC-1) for macrophages and some neutrophiles……. CD68 for histiocytes….

macrophages - Cd11b or f4/80, and neutrophils - RB6