Hello,
I've been (not so successfully) trying to perform basic experiments on dendritic cells to analyze the actions of a fatty acid. I've noticed there are numerous methods to do this and it seems that many people use completely different methods to arise at the same population. I've tried a variety of these methods and have not obtained similar results.
For example, I first tried isolating BM cells (femur flush) and treated them with GM-CSF and IL4 at 20 ng/mL or 10 ng/mL for 5-10 days. I never reached a CD11c% > 60. I've tried taking the adherent cells. I've tried taking non-adherent cells, and now I'm going to try obtaining both. These methods are routinely published and groups get >90% with or without positive or negative selection with beads. It seems that 50% of papers will actually publish the flow results for the DCs before they use them.
Meanwhile Helft et al. 2015. Immnity 42 demonstrated that even CD11c+ cells are 50% macrophages and not DCs. These data were debated (Helft Immunity 2016, Guilliams Immunity 2015, Lutz Immunity 2016) but there still was never a general consensus. More recently, a paper published in J Immunol (Jin and Sprent 2018) demonstrated even newer methods that yielded greater amount of DCs by using less cells and adding IL4 late during culture.
For someone new in the DC field this is very confusing and it seems that there is no tried and true method to isolate DCs. Is the CD11c+ count all that matters? How many markers do I need to show are active upon stimulation? Is CD80/CD86 enough? Do I need to show CD115, CD64, CD24? If so, this is barely ever done. Many groups also just say "We isolated DCs as done previously". This is great except it cites papers from nearly 20 years ago.
I am sorry for the long-winded questions but any help/direction would be very much appreciated!
Best,
Here are the papers I mentioned:
Article Alive but confused: heterogeneity of CD11c(+) MHC class II(+...
Article Still Alive and Kicking: In-Vitro-Generated GM-CSF Dendritic Cells!
Article GM-CSF Mouse Bone Marrow Cultures Comprise a Heterogeneous P...
Article A death notice for in-vitro-generated GM-CSF dendritic cells?
http://www.jimmunol.org/content/early/2018/10/12/jimmunol.1800031 (Jin and Sprent)
Dear Gregory,
Don´t lose heart. I suppose nobody can get you an universal-recipe for you. When we start to work with DC´s my counterpart and I have no experiencs with this. We clerify step for step all qustions we need for our speciel aim, e.g. Medium, conc. of Cytokins , we start with CD80, CD86, HLA-Dr, CD1a, CD14 and so on. Timepoints for best differciation and so on. I don´t know the circumstances in your lab wether the time you need for your tests, but the best way to sucess is to find out step for step in view of your aim what is the right way. For exampel our expiriences are if you want to detect Cytokines it is better to use serumfree Medium.
Jochem
Have you contacted the authors from the papers you mentioned? They may be most willing to help you.
First the easy answer... Yes, there are a lot of slight differences in DC differentiation from bone marrow. With IL-4, it pushes for proliferation, but it can potentially (as some have published) to skew the population to a difference sub type. I don't know why people don't do a positive selection after differentiation because there are other cell types in the non-adherent population. And for immature DCs, I would suggest you only take the non-adherent population after differentiation and do a positive selection for CD11c. If you don't want to, the only other way to "purify" you population is do let them adhere and wash away the non-adherent cells.
And the question whether CD11c is macrophages or dendritic cells. Truthfully, in mice, yes, we tend to use CD11c as THE marker. But even I can tell you that in practice, I have seen cells change markers from DCs to Macs. They usually odn't go back the other way. As for how many markers... this is tough, because macrophages almost always express the same markers (including CD80/86) and with the subset issues, the markers are confusing. Although... you can try DC-SIGN, which I'm pretty sure is resticted to DCs even in the mouse...
Probably the only way to get a "pure" population is to force maturation after differentiation. Usually once the immature DCs are activated, they don't tend to revert. But if you are looking at how your fatty acids mature DCs... then this won't work for you. One good way is actually by morphology. DCs and Macs are very different in cell morphology under the microscope.
So... what is the answer??? Work with what you can. Depending on what you are trying to answer in your experiments, you can probably use flow to only look at the effect on DCs.
Hi Gregory,
according to Shen Hang Wang cell Morphology is different as well as in FACS without staining (FSC/SSC). After isolation with magneto-beads from MACS and seeding in 12-well plates (RPMI 1640) using GM-CSF and IL4 for 5 days we observed that a part of the cells switched between adhärent and non-adhärent. Seperate analysis show exact the them pattern of CD-Marker. Sometimes I use only GM-CSF for 5 days and I observe the cells became more morphology like macrophagen und the cells express much more CD91 than DC´s. CD80 produce very different results in dependency of the clon we use for staining.
Jochem
Thank you everyone! It seems that it comes down to sticking to a method and taking care to characterize that model well. Another paper just came out that illustrates this conversation well:
https://www.nature.com/articles/s41590-019-0313-5
It appears that after stimulation with GM-CSF they again confirm three populations of cells (2 macs, 1 DC) characterized with CD11c, CD11b, MHCII (classical phenotyping). One population can activate inflammasome signaling while the other cannot. This could possibly be confusing without separating these cells into their populations.
I think I am going to try using GM-CSF in culture and then use negative selection (CD3, B220, CD49b, Ly6g, CD115) to isolate DCs and characterize with CD11c, CD11b, MHCII, and CD135 (like both papers above).
Best,
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