Monocytes are divided into 3 subsets based on their expression of cd14 and cd16 by flow cytometry; Classical(CD14++CD16-), Intermediate(CD14++CD16+) and Non-classical(CD14+CD16++). When I gate on the CD14+ cells in order to define the proportions of each subset I will often have a population in the lower left corner which are CD14low and CD16low. Does anyone know what type of cells this population might be?
See
Time to abandon dogma: CD14 is expressed by non-myeloid lineage cells
Immunology and Cell Biology (2005) 83, 462–467; doi:10.1111/j.1440-1711.2005.01370.x
Dear Eóin O'Brien
for a proper answer to the question it would be important to see a dot plot of your CD14 CD16 staining. Can you upload this?
Please also provide a description of your staining strategy and gating strategy including all scatter and fluorescence parameters.
Yours
Loems
See
Time to abandon dogma: CD14 is expressed by non-myeloid lineage cells
Immunology and Cell Biology (2005) 83, 462–467; doi:10.1111/j.1440-1711.2005.01370.x
Hi,
Thank you for the responses. I have attached an example of the gating strategy which I am using. The population I am talking about is in the first CD14 vs CD16 graph and is labelled "I".
Many thanks,
Eóin
Dear Eóin O'Brien
this plot is very informative. The events you see in this area are a) spill over of CD14 and CD16 negative cells b) CD14low CD16negative cells. The latter cells also express CD1c and have been classified as CD1c dendritic cells. For a subsets of these DC a low level expression of CD14 has been described.
Your gating for the monocyte subsets looks fine.
Yours
Loems
Dear Loems,
Thank you very much for the response. It will be very helpful having that information for analysing my data.
Thank you,
Eóin
Dear Eóin O'Brien,
your first gate (H), using FSxSS, is also getting some cells that are not monocytes. Try using a polygonal gate instead of the square one and place it closer to the green area of the density plot, avoiding "contamination". You will probably see a decrease in the mentioned population (gate "I"), since it seems to express no CD14 to me (the levels are close to the background). However, if you perform gating close to the monocyte population and still observe some CD14 low cells, then probably those cells are CD1c positive (DC), as mentioned by Dr. Loems.
Yours Sincerely,
Raphael
I second the comment of Dr. Harris. The monocyte field would be so much further along if CD14 were not labelled as a monocyte maker by the early days of flow cytometry. Prior to forcing monocyte activation/development into arbitrary stages based upon a few markers, such as CD14 and CD16, the field should redo the studies of the 1990's but integrating more monocyte/macrophage specific or informative antigens, such as CD64, CD163, CD206, CD300 and more in a multiparameter fashion. Once done properly, the answer to your question will likely be revealed.
- Badges
- Science method