|Cell Line:||Human keratinocyte (hKT)|
Vulgar Name: Human
|Age/Ethinicy:||5 years/ White|
|Derivation:||Established from human foreskin|
|Applications:||In vitro Assays for Research and Industry|
Keratinocyte Basal Medium (KBM) supplemented with Keratinocyte Growth Medium (KGM)-Lonza
1- Remove and discard the culture medium.
2- Rinse the bottle briefly with 1x PBS solution to remove remnants from cellular metabolism
3- Add 2.0 to 3.0 mL trypsin-EDTA solution to the flask.
4- Observe the bottle under the inverted microscope until the cell layer is individualized and derelict (usually between 5 and 15 minutes). NOTE: In order to avoid the breakdown of the cells into clusters, the bottle should not be stirred until the effective action of the trypsin. The bottle can be placed at 37º C (optimum trypsin temperature) to optimize the process. If, during the expected time, the cells are individualized but still adhered, the bottle can be shaken moderately against the palm of the hands or flat and smooth surface.
5- Add DMEM medium supplemented with 10% FBS in volume proportional to the previously placed trypsin solution (2.0-3.0 mL).
6. Shake with the pipette the cell solution with trypsin and medium and transfer gently to a Falcon tube.
7- Remove an aliquot for counting in Neubauer's chamber.
8. Centrifuge the cell suspension.
9- Subculture: Plaquee the amount of cells already determined by previous protocol in new bottles.
NOTE: For more information on enzymatic dissociation and cell subculture, see the 12th chapter of R. Ian Freshney's book Culture of Animal Cells, 6th edition, published by Alan R. Liss, N.Y., 2010.
Medium Renewal: Every 2 to 3 days
|Culture Conditions:||Atmosphere: air 95% and carbon dioxide (CO2) 5%|
|Cryopreservation:||50% FBS +40% KBM + 10% DMSO (Dimethyl sulfoxide)|
|Thawing Frozen Cells:||
R. Ian Freshney's book Culture of Animal Cells, 6th edition, published by Alan R. Liss, N.Y., 2010.