Hi Sharad,

They are different. You should consider the floating cells as iDC. CD86 and CD40 are highly express in mDC. CD14 is not. Both of them should not be express in iDC. You should ckech. I am not quite sure if CD1a will help you in this experiments.

Good luck

Marcelo

Hey,

duriong the mature of DC out of monocytes, they lose the CD14 expression and became CD14-. The expression of CD86 is high in iDC. CD1a is diminuished in iDC but raise again when DC became mature. We also check CD80 and HLA-DR, CD11c high.

Greetz,

Isabelle

You should see a loss of CD14 on floating cells, Typically MHC II should be dim to high and be up regulated strongly by activation stimuli together with CD86, CD80 and CD40. Of course you can also check activation by the expression of DC-LAMP which only appears in activated MODCs.

Hello,

iDC usually have a higher CD209 and ILT3 expression, compared to mDC.

Earlier, we compared the non-adherent and adherent fraction of iDC, and found that the floating fraction have somewhat higher MHCII and CD86 expression and somewhat lower CD1a expression, but the differences were always much smaller then between two PBMC donors. Furthermore, you will always have variability between the cells in both floating and adherent fractions, such as in phagocytic capacity and phenotype. Some mDC, such as the ones matured with poly I:C, immediately adhere after stimuli and may stay adherent although mature in phenotype and functions. Therefore, I recommend you to collect as many cells as possible per DC differentiation (maybe you could use low adherent plates for purified CD14+ monocites) and to use more than 2 donors per experiment, which will provide both enough cells per experiment and variability between donors.

Best luck,

Fully agree with Isabelle,

Hi Sharad,

iDC are CD14-, CD1a+, CD11c+, CD40+, HLADR+, CD86 low, CD83-, CD80lo/-.

mDC (LPS matured) are like iDC but CD83/CD86hi, CD80+.

I agree with those answers. Anyway, I think that performing an experiment marking with MHCII, CD11c and CD86 should be enough to distinguish iDCs from mDCs.

You should consider to stain the floating cells (immature MDDCs) with DC-SIGN which is a good marker for MDDCs even if it's expression slightly decreases after maturation with appropriate TLR agonists such as LPS (Like DC-LAMP, CD83 is a good maturation marker for human MDDCs). You may also extend a bit you differentiation time to 6 or even 7 days to get a stable "immature" phenotype as mentioned above by Matthias.

The adherent cells are probably macrophages that differentiated from the monocytes, whereas the floating cells are going to be your immature DC (iDC). iDC will be CD14-, CD83low or neg, and CD86-. My advice would be to do a stain for MHC class II and CD14. iDC should be positive for class II and neg for CD14. CD83 and CD86 will be most useful for staining when you have mature DC (mDC), which you do not have yet based on your described methods. Hope this helps!

Hi Sharad,

I agree with Sergej and Franck. You should use DC-Sign (CD209) as marker for immature and mature monocyte-derived DCs (moDCs). I get always a very strong signal with DC-Sign. It is also expressend on some macrophages, but that should not be a problem, if you use enough IL-4 in your moDC generation and extend your differentiation time to 7 days (see attachment).

In addition CD1c (BDCA-1) is also high expressed on moDC. Furthermore I use CD14 and CD1a as control of differentiation (moDC: DC14-, CD1a+dim) and CD80, as well as CD83 as maturation markers (immature moDC: CD80low, CD83+ / mature moDC: CD80high, CD83high). You can also use CD86. Mature moDC have a higher CD80 level as immature moDC. However, I think that it isn't a very good maturation marker, because it is already highly expressend in immature moDCs.

I use the adhenerent and the non-adherent moDCs in my experiments. Both are CD14- and DC-Sign+. They should be proper immature moDCs. As Sergej metionend, you certainly will see more variations between different donors, as between adherent and non-adherent moDCs.

I hope that my comments will help you a little bit.

Good luck with your experiments!

Thank you Very Much guys. I will certainly implement the information provided and let you know of the results. One more question , Inorder to MAture the DC cells I was going to incubate them with 1ug/ml of LPS (only- I will not be using any other maturation cocktails) for 24 hrs. In this case should I add GMCSF and IL4 in the medium with the LPS or the medium should contain only LPS and is LPS on its own sufficient for maturation or are other cytokines (TNF-alpha) essential.

LPS w/o Cytokines is fine...

Sharad-

For what purpose are you using the DC? LPS alone will put your DC in a semi-mature to mature state, depending on the health of the donor, but to get a really nice, mature DC I would recommend using additional cytokines, such as IFNg, TNFa, IL-1b, etc. The objective of your work will severely determine the type of maturation cocktail you should use. Additionally, I always wash my immature DC extensively before maturing so that all of the IL-4 is removed. For my purposes, I want type-1 polarized DC, and IL-4 drastically inhibits the differentiation of an iDC into a type-1 polarized DC. So the point is that your DC will be fine without GM-CSF and IL-4 while you are maturing. I would also recommend letting them mature for 48h, especially if you are using LPS. This will give you a higher percent of DC that are, in fact, mature, as opposed to maybe 50% of them being mature.

Dear Kellie

Thank you for your response. I am actually going to impulse iDC with apoptotic cells and then see the effect on maturity of DC by exposing them to LPS for 24h and then check for maturity using markers in FACS.

When you wash your iDCs extensively how do you do that ? When I tried to wash my cells at 200 g for 10 min, I also end up with debris. Is it possible to use a lesser time and also not loose the DC cells ?

S 100 protein is also stain the iDC s

ıf you obtain iDC from skin you can be floated suction blister roof in culture medium. Langerhans cells migrate to the medium and for the maturity of these cells you can add GMCSF to medium