3T3-J2 is a subclone of the original mouse embryonic fibroblast line; 3T3-Swiss Albino. After irradiation, these cells are commonly used as a feeder layer for growth of human keratinocytes.
Keratinocyte stem cells reside in the basal layer of the epidermis, which is the lowest layer of the stratified epithelia. These cells divide to give rise to transient amplifying cells which divide further, and differentiate, as they move upwards in the epidermis. The differentiating cells produce compounds and other proteins which are critical to the integrity of the outermost layer of the skin, the stratum corneum. The keratinocytes in the stratum corneum are dead squamous cells that are no longer multiplying. Once keratinocytes reach the corneum, they are said to be keratinazed, or cornified, creating the tough outer layer of skin.
From the laboratory of Howard Green, MD, Harvard University.
|Product Type:||Cell Line|
|Cell Type:||Embryonic mouse fibroblasts|
|Source:||12-13 days mouse embryos|
|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 penn/strep , 2mM glutamine and 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|
When thawing these cells, do not centrifuge, as this can lead to a poor recovery rate. The providing laboratory recommends:
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. Finally, when the cells have been passed 12 to 13 times, go back to a low passage frozen stock.
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