This cell line is a suitable transfection host.

The cells retain the ability to undergo squamous differentiation in response to serum, and can be used to screen chemical and biological agents for ability to induce or affect differentiation and/or carcinogenesis.

These cells should be subcultured before reaching confluence since confluent cultures rapidly undergo squamous terminal differentiation. Volumes used in this protocol are for 75 cm² flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.xa0Corning^® T-75 flasks (catalog #430641) are recommended for subculturing this product.

1. Remove and discard culture medium.
2. Add 2.0 to 3.0 mL of 0.25% Trypsin - 0.53mM EDTA solution containing 0.5% polyvinylpyrrolidone (PVP) to flask and observe cells under an inverted microscope until cell layer is dispersed (usually with 5 to 10 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach.xa0 Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
3. xa0Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.xa0
4. Transfer cell suspension to centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes
5. Discard supernatant and resuspend cells in fresh growth medium. Inoculate new flasks at 1500 to 3000 cells per cm².xa0 The culture flasks used should be pre-coated with a mixture of 0.01mg/ml fibronectin, 0.03 mg/ml bovine collagen type I and 0.01 mg/mL bovine serum albumin dissolved in BEBM.xa0xa0
6. Place culture flasks in incubators at 37°C.

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.

1. Prepare a mixture of 0.01 mg/mL fibronectin, 0.03 mg/mL bovine collagen type I and 0.01 mg/mL bovine serum albumin (BSA) dissolved in culture medium. Store pre-prepared Coating Solution at 4°C in cold room for up to 3 months.xa0
2. For a growth area of 75 cm², add 4.5 mL of the fibronectin/collagen/BSA solution and rock gently to coat the entire surface.xa0
3. Incubate the freshly coated vessel(s) in a 37°C incubator overnight (it is preferable to use tissue culture vessels with tightened, plug-seal caps to prevent evaporation during the coating process).xa0
4. Store coated flasks with solution at room temperature, light protected, up to 1 month. Suction off solution before plating cells.

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Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.