So for the differentiated Macs and DCs, you can probably use CD11c for DCs and CD68 for macs (the CD68 is intracellular so...plan accordingly if you are staining cell cultures, but they work under normal histological staining protocols for FFPE tissues).

Since you said that you are also working with epithelial cells, I'm assuming that your primary lung epithelium is also a culture and not actual lung tissue. If the latter, CD11c probably won't work since alveolar macrophages do express some DC markers.

Hi Shen-An Hwang,

Thanks for your reply.

Okay thank you! No, indeed it is a culture. So I can use CD11c and CD68? For the CD68, as I want to do immunofluorescence to image my immune cells, will it be a problem that it is intracellulare to target it with a primary and a secondary fluorescent antibody?

And I can use CD14 as marker of monocytes as the expression will be absent from Macs and DCs, right?

I saw that CD45 is a general marker for leukocytes and so I could use it to image my immune cells on the epithelial cells.

Yes, CD45 is the pan leukocyte marker.

CD14...I'm pretty sure it's still on macrophages.

As for CD68, technically yes, you can use a secondary antibody...although on second thought...

You can also try DC-SIGN for DCs... The problem is that it's quite difficult to use only one marker to differentiate between monocytes, DCs, and macrophages... (much easier to do the analysis using flow, since I can use multiple markers) (especially with the subtypes since most macrophage and DC cultures are quite heterogeneous, unless you have a strong culture condition to push them one way or another...)

I think what you would need to do is to see which combination works best in you in your system.

Although since this is cell culture you might be able to tell the differences by morphology?

Hopefully someone else will have an idea.

Okay thank you!

Yes I'll be able to differentiate them by morphology hopefully.

I agree with Shen-An. First of all, it is important to know that the lung contains around 20% of dendritic cells comprising about ten subpopulations whose alveolar macrophages and type II pneumocytes are closely related. Pneumocytes of type II alone represent 10% of the total cells of lung tissue, and are characterized by the SP1 marker (for surfactant) and CD208 (DClamp) which is a marker of activated DCs. Then we have a multitude of DCs characterized by CD204,205,206,207,209 polyforms, 303,304, ASGPR. But these different subpopulations of DCs and / or macrophages are very unstable in primary culture and disappear very quickly, to let the fibroblasts take over and invade the culture. It is therefore very important to use cytokine cocktails (TNFa, IL3, IL4, IL10, GM-CSF, TGFb ....) to preserve these different subpopulations. It is also important to perform cell sorting so as not to be invaded by unwanted fibroblasts. If you are looking for images to convince you of all this you can look at the banner of the site www.dendritics.net

Hi Fabio, since you are isolating monocytes from PBMC and differentiate them in vitro, I strongly suggest you to confirm their phenotype by FACS first and them proceed with the IF. Consider that already by scatter plot the macrophages appear very different (hi ssc) from monocytes and DC (low ssc) and add as well some surface marker, however the population of mononuclear phagocytes in the lung are very different from the in-vitro differentiated, at lest in terms of surface marker. I did a similar study on human kidney mononuclear phagocyte where you can find some of the marker used for distinguish the three population, also in tissue histological section.

Article The Phenotypic Characterization of the Human Renal Mononucle...

Thanks Jean-Jacques Pin

!

Hi Dario Armando Leone,

Thanks a lot!!