|Cell Type:||Embryonic mouse fibroblasts|
|Subculturing:||Subculture the cells once they reach 60-80% confluency. Split the cells at a density of between 3.5-5 x 103 cells/cm2. Feed every 2 to 3 days with complete medium.|
|Growth Conditions:||Cells grow in 10% CO2 at 37C in a humidified incubator. Media: DMEM supplemented with 10% bovine calf serum. DO NOT use fetal bovine serum. Growth in fetal bovine serum affects the growth potential of these cells. Bovine calf serum should be iron supplemented and NOT gamma irradiated or heat inactivated (Recommended serum: GE Healthcare Iron-Supplemented BVN CLF 500ml; Cat.SH30072.03).|
|Cryopreservation:||DMEM with 10% bovine calf serum containing 10% sterile DMSO|
|Source:||12-13 days mouse embryos|
When thawing these cells, do not centrifuge, as this can lead to a poor recovery rate.
It is very important not to let the cells get confluent or they will lose their contact inhibition. This will affect their ability to support cells when used as a feeder layer.
We always use preconfluent or just confluent flasks for our feeder layer when growing keratinocytes. * If you continuously pass feeders from post-confluent flasks, some of the cells will begin to lose their contact inhibition. It is normal to notice a slight rise in the saturation density of the flask as you increase passage number, but the maximum number of cells you should get from a just confluent 150 cm2 is about 5-6 x 106. If you are getting on the order of 8 x 106 cells or higher, then your feeders are not being passed properly. This can affect the quality of your keratinocyte culture, so these guidelines should be strictly adhered to.
- Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell. 1975 Nov;6(3):331-43.
- Allen-Hoffmann BL, Rheinwald JG. Polycyclic aromatic hydrocarbon mutagenesis of human epidermal keratinocytes in culture.Proc Natl Acad Sci U S A. 1984 Dec;81(24):7802-6.
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