Hi all,
I am analyzing the myeloid cell populations in pancreas of NOD (non-obese diabetic) mice.
My gating strategy for now is as follows:
Gate for live CD45+ cells --> Select CD11b+ myeloid cells --> Gate out the Ly6G+ neutrophils, select the rest for further sub-gating --> Gate out Ly6C+CD14+ monocytes, select the rest --> On a plot with F4/80 vs CD11c, I get 4 populations:
- F4/80+CD11c = Macrophages
- F4/80-CD11+ = Dendritic cells
- F4/80-CD11c- = Monocyte-derived macrophages?
- F4/80+CD11c+ = ??
Has anyone encountered that before when analyzing pancreas from NOD mice? Do you have any tips/suggestions?
Thanks in advance!
Alba
#flowcytometry #NODmice #pancreas #myeloid #macrophages
Hello Alba!
Well... there are a lot of controversial opinions about some markers of these two myeloid cells for non-lymphoid tissues and mainly in inflamed sites… The first question is that if your study is in a steady-state condition or in an inflammatory one. There are a lot of inflammatory cells expressing CD11c and F4/80 during inflammation which are not even DCs nor Macrophage. These cells are currently known as Inflammatory-derived cells. (See Guilliams M. et 2014 “Dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny.)The second task is if you are using CD64 to differ Macrophage and DCs only macrophages expresses CD64 . Are you only using F4/80 and CD11c in your flow cytometry panel?
There are DCs expressing F4/80 and Macrophages expressing CD11c. So, it is very difficult to analyses these cell populations with no cross contamination only using these markers
See Guilliams M. et 2016 “Unsupervised High-Dimensional Analysis Aligns Dendritic Cells across Tissues and Species”.
This is the best strategy to differentiate DCs and Macrophages that I know. They considered CD64+F4/80+ as macrophages and CD11c+ cells DCs.
I do recommend you to use CD64 mainly in non-lymphoid tissues
I hope it helps you
Agree to Fernando. Second issue which you should take into account is aggregation of cells. This may be source of many artifacts, especially if you see double-positive subpopulations of cells.
Thanks a lot for your contributions Fernando Sulczewski and Dmitry B. Kazansky !
I do exclude duplets using FSC-H and FSC-A, so I believe that I am looking only at singlets.
Regarding the extra marker to differentiate DCs and macrophages, I tried using an anti- I-A/I-E antibody but it did not work so well. I take your advice of including an anti-CD64.
Thanks again!
Hi Alba all comments are great. The guy you want to read on this is Pieter J. M. Leenen he had done some great characterization of myeloid lineage development in mice and some specifically in the NOD mouse highlighting the defects in many aspects of macrophage function. ALso David Serreze did a great deal of fantastic characterization of NOD macrophage DC defects.
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