Dendritic Cells (DCs) are highly motile immune cells and are the most powerful antigen presenting cells (APCs) of the mammalian immune system.1 DC’s act as messengers between the innate and the adaptive immune systems by continuously sampling their environment for antigens by means of endocytosis.1 Being responsible for the induction T-cell dependent immunity and tolerance, they are especially abundant in epithelia (skin and intestinal tract) and other tissues that frquently encounter invading pathogens.2 Currently, there are two known major dendritic cell types in humans: 1) lymphoid dendritic cells derived from plasmacytoid cells in the blood and 2) myeloid dendritic cells derived from myeloid precursor cells such as peripheral blood monocytes (PBMCs) or CD34+ hematopoietic progenitor cells from the bone marrow. 1 DCs develop by means of a maturation process induced by exogenous or endogenous stimuli ( Fig.1). More recently, dendritic cells have been used for their potential development of cancer immunotherapies3 as well as the treatment of autoimmune diseases.4 Since DCs also play an essential role in the setting of HIV infection5 and the pathogenesis of several other viruses, they have significance as a therapeutical target.
PromoCell’s Dendritic Cell Generation Media (C-28050) allows for the efficient and reliable in vitro differentiation of human monocytes (hMo) into immature as well as fully mature CD83+/CD14- dendritic cells. The media can be used for the differentiation of cryopreserved purified monocytes into moDCs. Futhermore, the media may also be used with freshly isolated monocytes and MNCs when combined with the PromoCell Monocyte Attachment Medium (C-28051). The chemically defined and xeno-free PromoCell Dendritic Cell Generation Medium DXF (C-28052) is formualted exclusively with synthetic, recombinant or plant-sourced materials and is recommended for use with freshly isolated monocytes or mononuclear cells (MNCs).
Figure 1. Differentiation of human PBMC and CD34+ derived monocytes into mature CD83+/CD14-dendritic cells.
Protocol A: Generation of moDCs from freshly isolated cells using DC Generation Medium DXF
For Generation of moDCs from freshly isolated peripheral blood Monocytes or Mononuclear Cells, PromoCell recommends the use of the Dendritic Cell Generation Medium DXF (C-28052). Refer to protocol A for details.
Alternatively, the Dendritic Cell Generation Medium (C-28050) in combination with the Monocyte Attachment Medium (C-28051) may be used. See protocol B for detailed procedure.
Protocol B: Generation of moDCs from freshly isolated cells using DC Generation Medium and Monocyte Attachment Medium
Protocol C: Generation of moDCs from Cryopreserved Cells
For Generation of moDCs from cryopreserved peripheral blood Monocytes, PromoCell recommends the use of the Dendritic Cell Generation Medium (C-28050).
Figure 2. FACS analysis of mature monocyte derived human dendritic cells. Mature dendritic cell markers include high expression of CD83 and low expression of CD14. A. Flow-cytometry analysis of day 8 mature moDCs generated in the PromoCell DC Generation Medium are negative for the immature marker CD14 and positive for the DC maturation marker CD83.
Figure 3. Morphology of immature and mature monocyte derived human dendritic cells. A) Day 6 immature monocyte-derived dendritic cells (moDCs). Note the large cytoplasmic, veil-like processes seen in adherent (center) as well as loosely attached (upper left) cells. B) Day 8 mature monocyte-derived Dendritic Cell (moDC). Note the multiple dendrite-like structures arising from the surface of this non-adherent cell.