Can you please review/guide my marker selection? Ideally, I want to measure T cells, B cells, NKs, DCs, Monocytes, and Macrophages. I am having difficulties separating out DCs, Mono and Macro. Perhaps this is not possible with only 7 markers... Should I substitute CD11c for HLA-DR or CD16 for CD68?? Any input is very much appreciated. I have come up with the following:
CD11c, CD3, CD19, CD14, CD45, CD68, CD56 (8th channel is for live/dead)
T cells: CD45+ CD3+
B cells: CD45+ CD19+
NK: CD45+ CD56+ CD3-
NKT: CD45+ CD56+, CD3+
DCs: CD45+ CD11c+ ?
Monocyte: CD45+ CD14+
Macrophage: CD45+ CD14low/- CD68+
Many thanks,
In fact, almost all DC markers are missing.
You need to choose from the many known (but also lesser known) DCs markers, to achieve your goals.
The most common (but least useful) markers: CD1a, CD1c, CD141, CD303, for blood.
CD1a, CD201, CD204, CD205, CD206, CD207, CD208 and CD209 for the spleen.
And for tumors: everything that can mark the DCs: but above all CD206-207-208-209-303-304-367, and all the TLRs.
We are currently doing the same, all these markers give edifying information. It is very heavy but unavoidable, and in addition it varies from one tumor model to the other.
Good luck if you find the solution with 7 colors.
Not sure if that helps but you can have a look at these recent papers concerning the antigenic definition of human myeloid cells.
Also, even if you are restricted to 8 colors, you can still detect more than 8 different markers in some cases by exploiting the FSC-A and SSC-A plot and if you are positive that the two markers are not expressed by the same cell type. For instance you could use FITC for both CD3 and CD14, and distinguish them very clearly based on FSC-A SSC-A. It works well in some cases (maybe less well on frozen / fixed / permeabilized cells). If you have fresh cells, it's worth trying though. There is another example of such a strategy in the paper by Frischmann et al. (see link below) where they detected 9 markers using 4 colors...
Good luck ! :)
https://www.ncbi.nlm.nih.gov/pubmed/27637149
https://www.ncbi.nlm.nih.gov/pubmed/28473638
https://www.ncbi.nlm.nih.gov/pubmed/28428369
https://www.ncbi.nlm.nih.gov/pubmed/16496423
In fact, almost all DC markers are missing.
You need to choose from the many known (but also lesser known) DCs markers, to achieve your goals.
The most common (but least useful) markers: CD1a, CD1c, CD141, CD303, for blood.
CD1a, CD201, CD204, CD205, CD206, CD207, CD208 and CD209 for the spleen.
And for tumors: everything that can mark the DCs: but above all CD206-207-208-209-303-304-367, and all the TLRs.
We are currently doing the same, all these markers give edifying information. It is very heavy but unavoidable, and in addition it varies from one tumor model to the other.
Good luck if you find the solution with 7 colors.
Also note that CD68, is a poor surface marker. It works best with intracytoplasmic staining.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology