Which DC medium does PromoCell recommend for freshly isolated MNC or monocytes, which one for cryopreserved monocytes?

The PromoCell DC Generation Media have been developed for the easy and efficient generation of immature as well as fully mature myeloid dendritic cells from peripheral blood monocytes. For freshly isolated mononuclear cells (MNC) and monocytes we recommend our DC Generation Medium XF (C-28052), for cryopreserved monocytes our DC Generation Medium (C-28050). When using DC Generation Medium (C-28050) with fresh MNC, the Monocyte Attachment Medium (C-28051) is needed in a first step for efficient adherence of the monocyte fraction. Please see Instruction Manual, Application Note and the graph below for further details.

Does PromoCell recommend a specific plasticware to use for the generation of dendritic cells from peripheral blood monocytes?

Usually, we do not recommend specific plasticware but we have found that with PromoCell’s Dendritic Cell and Macrophage Generation Media, choice of plasticware can have a lot of an influence. For our Dendritic Cell Generation Media we recommend to use tissue culture vessels from BD Falcon TM .

Is there is a difference in the percentage of differentiated DCs depending on whether we start with cryopreserved or fresh isolated monocytes?

There is no significant difference in the percentage of cells that will differentiate into Dendritic Cells. But when using cryopreserved cells, the initial cell loss will be higher compared to when fresh cells are used. i.e. the final number of differentiated cells that can be expected will be higher with fresh cells as a starting material due to lower cell death rate.

We use Macrophage Generation Media from PromoCell to get differentiated macrophages from peripheral blood monocytes. The monocytes are isolated by culturing PBMCs in Monocyte Attachment Media (also from PromoCell) for 1.5 h. Would it be ok if the attachme

It is not recommended to leave the blood cells in the Monocyte Attachment Medium for longer than 1.5-2 hrs. The medium was developed for (short-term) attachment of the monocytes and does not provide nutrients for a longer time period. Leaving the cells in Monocyte Attachment Medium for a longer time or even overnight will induce apoptosis and lead to the loss of the cells. If necessary, you can reduce the incubation time to 1 hr. In this case, it is advisable to equilibrate the media in the incubator before so that you can immediately and directly add the appropriate amount of PBMC suspension.

What are the advantages of monocyte enrichment by adherence selection using your Monocyte Attachment Media (C-28051)?

This method produces large numbers of adherent monocytes in only 1.5 h. If the washing steps are properly performed, 80-90% purity can be expected. The attached cells are “untouched”, since no binding of magnetic microbeads has occurred. This also excludes phagocytosis of the microbeads by the monocytes, an event which is unfavorable with regard to cellular health. In addition, the adherence method is time-saving and cost-effective.

How can I test whether my cells are infected with mycoplasma?

Several different methods for the detection of mycoplasmas have been described, like e.g., cultures on agar, in liquid or semi-solid media, staining with DAPI, mycoplasma-specific antibodies, biochemical methods, and PCR-based assays. PCR-based detection is very sensitive, detects all mycoplasma species that occur in cell cultures and is completed within 3-5 hours.

Can mycoplasma contamination be observed with the naked eye?

No, mycoplasma can only be observed through electron microscopy. For highly sensitive detection of mycoplasma contamination, we recommend the use of PCR-based mycoplasma tests.

How does PromoCell determine the phototype of skin tissue donors?

We use a classification system similar but not identical to the Fitzpatrick Skin Classification. The Fitzpatrick classification has six different categories (phototypes I-VI) which correlate with the level of skin pigmentation (melanin) and sunburn following sun exposure. Fitzpatrick I corresponds with the lightest of skin complexions, while Fitzpatrick VI corresponds with the darkest skin. I: Pale white skin, blue/hazel eyes, blond/red hair, always burns, does not tan II: Fair skin, blue eyes, burns easily, tans poorly III: Darker white skin, tans after initial burn IV: Light brown skin, burns minimally, tans easily V: Brown skin, rarely burns, tans darkly easily VI: Dark brown or black skin, never burns, always tans darkly At PromoCell, we have knowledge of the patients’ skin color (white, brown or black skin), color of eyes and hair, but we don't have any details about the burning/tanning abilities. We therefore classify our tissue donors as follows: Light (comprising phototypes I and II) Moderate (comprising phototypes III and IV) Dark (comprising phototypes V and VI) Information on the phototype is available for most cell lots isolated from juvenile or adult skin.

Is it possible to carry out the macrophage differentiation in either a 96- or 384-well plate, so long as the volumes are adjusted accordingly? If not, is it possible for cells to be detached and re-plated in the desired culture plate?

a) We usually perform macrophage differentiation in T75 flasks and 6-well plates. We haven't tested differentiation in smaller formats. But we assume it will be problematic to thoroughly wash the surface of the wells to remove non-adherent cells after the attachment phase. b) Detachment of the mature macrophages is possible but re-attachment can lead to significant cell loss (30-50%). Please also keep in mind that working in 96/384 well-format has some inherent drawbacks (e.g., evaporation of media, dry wells, etc.).

Do you know in what media the monocyte-derived DCs can be maintained in culture after the differentiation is completed?

You should use complete DC Generation Medium/DC Generation Medium XF (with all the cytokines). As cells are metabolically active, media should be changed every 3 days. We have observed that the dendritic cell phenotype remains stable for up to 7 days.

Why is Human Serum Albumin (HSA) added to the PBS wash after detachment of macrophages with Macrophage Detachment Solution?

The Macrophage Detachment Solution (C-41330) directly affects the cell membrane. HSA in the Wash Buffer supports regeneration of the cell membrane and protects the cells during the critical phase directly after detachment from detrimental effects.

Are the HDMEC pre-screened (C-12215) derived from juvenile foreskin or from adult skin?

Our HDMEC pre-screened cells are isolated from juvenile foreskin.

Technical library - RESOURCES

We use a classification system similar but not identical to the Fitzpatrick Skin Classification. The Fitzpatrick classification has six different categories (phototypes I-VI) which correlate with the level of skin pigmentation (melanin) and sunburn following sun exposure. Fitzpatrick I corresponds with the lightest of skin complexions, while Fitzpatrick VI corresponds with the darkest skin.

I: Pale white skin, blue/hazel eyes, blond/red hair, always burns, does not tan
II: Fair skin, blue eyes, burns easily, tans poorly
III: Darker white skin, tans after initial burn
IV: Light brown skin, burns minimally, tans easily
V: Brown skin, rarely burns, tans darkly easily
VI: Dark brown or black skin, never burns, always tans darkly

At PromoCell, we have knowledge of the patients’ skin color (white, brown or black skin), color of eyes and hair, but we don't have any details about the burning/tanning abilities. We therefore classify our tissue donors as follows:

Light (comprising phototypes I and II)
Moderate (comprising phototypes III and IV)
Dark (comprising phototypes V and VI)

Information on the phototype is available for most cell lots isolated from juvenile or adult skin.