I would suggest positive selection of CD14 cells using the Miltenyi system. You should get 20-40 million cells from 100mls of blood. I usually culture around 8 million CD14 cells in 2 wells of a 6 well plate or 3-4 wells of a 12 well plate. I used RPMI medium with 10% AB serum. AB is better than FCS as you tend to get non-specific activation of the cells with FCS. The amount of GM + IL4 you are using seems fine. You should get some CD80 expression but I generally find CD83 expression is very low. You should also check CD86 and CD40. Expression of these can be increased by using 1mg/ml LPS and 25ng/ml TNFa overnight. The amount of attachment is variable but you should see small clusters of cells. When you harvest the cells/scrape them off many of the cells are in small clusters. You should get about a million DCs. Incubating the cells in 5mM EDTA, 1mg/ml BSA in HBSS should give you a single cell suspension.

Hello Lee Faulkner, 

Thank you very much for your answer. Recently, I have been able to differentiate monocytes to cells with dendritic cell morphology, which we are almost certain that they are immature DC. I observed that there are DC attached and others not. Reviewing papers, I found that immature DC are semi non-adherent, you can find them attached or non-attached, could you confirm this?

I know that FCS is not the best option (because non-specific presentation of bovine serum antigens by HLA), so the next step will be to use RPMI with 5-10% of human serum (we used a commercial human serum)... do you think is better to use the serum of the own peripheral blood donor? If so, how do you obtain this serum from the plasma (plasma obtained from Ficoll density gradient, which it is diluted)? Do you have to let it to coagulate or do you remove the fibrinogen? 

CD83 is very low when they are immature DC, but when they are maturated with TNF-a, GM, IL-4, IL-1b, IL-6, and PGE2, I suppose that CD83 will be high, as I have seen in papers. I am going to comprovate it in the following days, when I stimulate DC with the maturity cytokines set and analyse them by FACS. Regarding CD86 and CD40, I will take them into account as an alternative markers for DC, in addition to HLA-DR, CD83 and CD80. I will only use LPS (100-200 ng/mL) overnight after the tumor lysate loading in order to highly stimulate the DC. Finally, I will take into account how to harvest/scrape the cells and obtain a single cell suspension.

I have done some questions that I would like to obtain an answer from you or from another. Again, thank you very much with all the contributions and recommendations that you have done in this question. 

Hello Joan,

I answer you in English to be understood by the rest of ResearchGate community. I worked 5 years with human monocyte-derived dendritic cells. You can see a detailed protocol in my thesis (it is in Spanish but I suppose you speak it): https://www.researchgate.net/publication/287995004_Activacion_de_IL23A_y_represion_de_IL12A_en_la_respuesta_inmune_Th17 (pages 24-25) and for the rest of ResearchGate community is also available in English: "Valera I, Fernández N, Trinidad AG, Alonso S, Brown GD, Alonso A and Crespo MS (2008). J. Immunol. 180: 5727–5736".

In summary, when you perform Ficoll gradient density centrifugation, you obtain peripheral mononuclear cells (PBMCs) which are composed of monocytes and a lot of lymphocytes. In order to purify monocytes, we performed two additional special gradient density centrifugation (described in the references above) to separate them from lymphocytes and after that, we cultured these cells in RPMI + 10% heat inactivated FBS + 1% Glutamine during 2 hours. At that time, monocytes have adhered on the culture dish whereas the remaining lymphocytes are in the supernatant, so we replaced the medium to eliminate the remaining lymphocytes and to get only adhered monocytes with fresh medium. Then, this is the time to differentiate them to dendritic cells during 5 days by using GM-CSF (800 U/ml) and IL-4 (500 U/ml). The cytometry profile of these cells is described in my thesis and the paper referenced above.

After 5 days, we got non-adherent or semi-non-adherent immature dendritic cells ready for experiments. When they are stimulated by any stimulus, they became adherent mature dendritic cells with their characteristic prolongations. But you don't have to pre-stimulate immature dendritic cells to get mature dendritic cells for your experiments, you should stimulate immature dendritic cells directly with your desired stimuli.

I hope this response is useful for you and other researchers.

Thesis Activación de IL23A y represión de IL12A por metabolitos en ...

Commercial serum is OK. CD40 and CD86 expression will give you some idea of how mature your DCs are.

Hallo Joan,

first of all you don´t get CD83++ Dc´s if you use only Gm-CSF and Il-4.

If you want full matured cells you have to add something like LPS for example. I send the protocoll we used.

Jochem

Hello,

I want to differentiate THP-1 cells into immature dendritic cells to use in further experiments (APC maturation experiment) I am found many papers that use GM-CSF and IL-4, but I have a drought on whether the cells are attachment or suspension after differentiation in to immature dendritic cells. Any one can help with this please?