Technical Library

Problems in obtaining RNA with good yield and purity from mononuclear cells (hMNCs) are quite common. The reason for this is the large amount of free genomic DNA usually contained in MNC preparations. This DNA originates mostly from granulocytes which underwent lysis during the isolation of the MNC. The granulocytes are gone in the final MNC preparation; but their genomic DNA - released during cellular lysis - is still there "sticking" to the MNCs. Solution: Remove DNA prior to RNA purification by a DNase digestion step. Most commercial systems include the option for such a DNase digest.

Our MSC Growth Medium XF (Ready-to-use) provides a xeno-free culture system for human MSCs. It contains all growth factors and supplements except attachment- and spreading factors. Therefore; culture vessels must be precoated with 10 μg/ml human or bovine fibronectin. Protocol: Fibronectin coating Dilute the Fibronection Solution to 10 μg/ml final concentration in Dulbecco´s PBS w/o Calcium and Magnesium. Overlay the culture surface of your tissue culture vessel with an amount of the diluted Fibronectin Solution sufficient to effectively coat the complete surface. Be sure that the entire surface is covered. Place flasks on a level surface at RT for 60 min. Aspirate the excess Fibronectin Solution and use immediately or let air-dry the open vessel under a laminar flow bench. Unused vessels may be stored at 4°C for up to 2 weeks.

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.

Our DC Generation Medium XF (C-28052) has a xeno-free formulation. It provides a complete media system (ready-to-use all cytokines included) and shows efficient and reproducible in vitro maturation of moDCs from freshly isolated peripheral blood monocytes.

It takes 7-8 days to generate fully mature myeloid dendritic cells.

The Monocyte Attachment Medium allows for the efficient adherence selection (within 1 hr) of monocytes from freshly isolated mononuclear cells while maintaining optimal cell health. The Time-consuming and costly immunomagnetic purification of monocytes prior to DC generation is not necessary and can be skipped when using this medium.

• DC Base Medium (C-28053) consists of 250 ml Basal Medium + SupplementMix. It does not include cytokines and must therefore be supplemented according to the user's individual needs.

• DC Generation Medium (C-28050) is ready-to-use and consists of 250 ml Basal Medium + SupplementMix + cytokines.

• DC Base Medium XF (C-28054) consists of 250 ml Basal Medium + SupplementMix. It does not include cytokines and must therefore be supplemented according to the user's individual needs.

• DC Generation Medium XF (C-28052) is ready-to-use and consists of 250 ml Basal Medium + SupplementMix + appropriate cytokines.

Both; Base Medium XF and Generation Medium XF have a xeno-free formulation.

The pH of our Endothelial Cell Basal Media MV/MV2 is 7.4 ± 0.1.