Ker-CT

Overexpression of CDK4 and hTERT in neonatal foreskin keratinocyte induced a dramatic upregulation of p16INK4a and milder upregualtion of p53 and p21WAF1, which became unresponsive to UV irradiation.  Despite the high levels of these checkpoint factors, Ker-CT cells divide in an apparently normal regulated fashion, are able to respond to changes in calcium levels, retain the stem cell phenotype, and fully differentiate and stratify in organotypic culture RefRamirez R, et al. Bypass of telomere-dependent replicative senescence (M1) upon overexpression of Cdk4 in normal human epithelial cells. Oncogene 22(3): 433-44, 2003. PubMed: 12545164

1. Check all containers for leakage or breakage.
2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

1. Prepare a 25 cm² or a 75 cm² culture flask containing the recommended complete culture medium. Prior to the addition of the vial contents, the vessel containing the growth medium should be placed in the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6) and to avoid excessive alkalinity of the medium during recovery of the cells.
2. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 minutes).
3. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All operations from this point on should be carried out under strict aseptic conditions.
4. Thaw the frozen vial at 37°C and resuspend the cells in 5mL of complete growth medium.  Count the cells and seed at recommended seeding densities. DO NOT centrifuge after thawing to remove DMSO. 
5. Incubate the culture at 37°C in a suitable incubator.
6. Change to fresh medium after the cells attached, usually 6-12 hours later, to remove DMSO and FBS.

1. Remove and discard spent medium.
2. Briefly rinse with HBSS (ATCC 30-2213), 1 mL/25 cm² and discard rinse solution.
3. Add trypsin for primary cells (ATCC PCS-999-003), 1mL / 25cm².  Place at 37oC for 4-6 minutes, until 90% of the cells have detached.  
4. Rap flask gently to ensure cells are detached. Add 2% FBS in D-PBS, 1 mL/25cm² to neutralize trypsin. 
5. Centrifuge cells at 250 x g for 5 min at room temperature.
6. Remove supernatant. Resuspend pellet in 6.0 to 8.0 mL Complete Growth Medium.
7. Count cells, and seed 5.0 x 10³ to 8.0 x 10³ viable cells/cm² to new culture vessels.

As the cells become more confluent, increase the volume of media as follows: under 25% confluence feed cells 5 mL per 25 cm², 25-45% confluence then feed cells 7.5 mL per 25 cm², over 45% confluence then feed cells 10 mL per 25 cm².

The product is provided 'AS IS' and the viability of ATCC^® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

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