How should I best freeze normal human cells?

Short protocol: Trypsinize the cells as usual Centrifuge and resuspend in suitable cold freezing medium at a density of 1-4 x 10 6 cells/ml Cool down the cells slowly to -80°C (approx. -1°C per min). We recommend to use "Mr. Frosty" from Nalge or "CoolCell" from Biocision, which both provide gradual and controlled cooling rates when placed in a -80°C freezer overnight. Transfer the vials into liquid nitrogen for long-term storage

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Yes, it is possible to transfect our normal human cells. In general, primary and normal cells are much harder to transfect than immortalized cell lines. Apart from the cell type, successful transfection also depends on the culture's age and density at transfection, the vector used, the purity of the nucleic acids, the composition of the transfection medium, and the experimental conditions.

Precoating of culture vessels with ECM proteins does not have adverse effects on the cells but has been reported to influence the cellular expression pattern. It is therefore recommended to use the same culture conditions, e.g. fibronectin-, collagen-, or gelatin-coating for a whole set of experiments to be able to compare the results.

PromoCell generally culture their HOB on uncoated tissue culture dishes. It is possible however to grow them on collagen type I- or fibronectin-coated dishes as well. Please note: The type of extracellular matrix used may influence the expression of certain genes (e.g. integrins) and thereby affect cellular metabolism. Therefore, we recommend to always use the same type of coating matrix for a whole set of experiments.

If you are growing our NHEK in PromoCell Keratinocyte Growth Medium 2 (C-20011) or Growth Medium 3 (C-20021), you don't need any feeder cells. The cells will grow as a monolayer in conventional tissue culture flasks.

PromoCell guarantee > 15 PD for their HUVEC. The number of passages that can be performed, depends on the dilution factor used during subculture. If you split the cells 1:4, they can perform about 2 population doublings per passage which means that they can be cultured for at least 6-8 passages.

Our MSC Growth Medium XF provides a xeno-free culture system for human MSCs. It contains all growth factors and supplements except attachment- and spreading factors.

Therefore, culture vessels to be used with Mesenchymal Stem Cell Growth Medium XF must be precoated either with 1 μg/cm2 human fibronectin or 0.5 μg/cm2 human vitronectin according to the instruction manual of the manufacturer.

Alternatively, bovine fibronectin may be used.

No, it is not necessary to use coated flasks, therefore we don´t recommend their usage in the Instruction Manual. However, for special applications, some of our customers use collagen-coated dishes.

Yes, our MSC Growth Medium XF (# C-28019) contains phenol red. The concentration is confidential.

But we also supply a phenol red-free variant: Mesenchymal Stem Cell Growth Medium XF, prf (# C-28018).

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.

Short protocol:

Trypsinize the cells as usual
Centrifuge and resuspend in suitable cold freezing medium at a density of 1-4 x 10⁶ cells/ml
Cool down the cells slowly to -80°C (approx. -1°C per min). We recommend to use "Mr. Frosty" from Nalge or "CoolCell" from Biocision, which both provide gradual and controlled cooling rates when placed in a -80°C freezer overnight.
Transfer the vials into liquid nitrogen for long-term storage

PromoCell HUVECs are freshly isolated from umbilical veins. They are cryopreserved at the end of primary culture. After revival, they can be propagated for at least 15 doublings and will senesce eventually. HUV-EC-C from ATCC is a hypodiploid human cell line of endothelial origin (umbilical vein). The modal chromosome number is 45 occurring in 72% of cells counted. The rate of polyploid cells is 15.8%. The cells have a life expectancy of 50 to 60 population doublings. This indicates that the cells are no longer "normal cells" but have undergone some degree of transformation.

Freshly isolated cells that are plated in a tissue culture vessel for the first time are named primary cells or primary culture (corresponding to P0). As soon as they have been subcultured, they should correctly be termed normal cells (> P1).