UACC-732

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.

The base medium for this cell line is ATCC-formulated Leibovitz's L-15 Medium, Catalog No. 30-2008. To make the complete growth medium, add the following components to the base medium:

• fetal bovine serum to a final concentration of 5%
• 0.01 mg/ml transferrin (final conc.)
• 0.01 mg/ml insulin (final conc.)
• 5 µg/mL (55 U/ml) catalase (final conc.)
• 3.6 µg/mL (0.01 mM) hydrocortisone (final conc.)
• 70 µg/mL (0.5mM) o-phosphoethanolamine (final conc.)
• 10 ng/ml human recombinant epidermal growth factor (EGF) (final conc.)
• 3 ng/ml (0.01 µM) estradiol (final conc.)
• 0.8 ng/ml (1 pM) Na-L-thyroxine (final conc.)
• 23 µg/mL (0.2mM) proline (final conc.)
• extra 2 mM glutamine

1. 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).
2. 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 of the operations from this point on should be carried out under strict aseptic conditions.
3. It is recommended that the cryoprotective agent be removed immediately.  Centrifuge the cell suspension at approximately 125 x g for 5 to 10 minutes.  Discard the supernatant and resuspend the cell pellet in an appropriate amount of fresh growth medium. 
4. Transfer the cell pellet to an appropriate size vessel.  It is important to avoid excessive alkalinity of the medium during recovery of the cells.  It is suggested that, prior to the addition of the vial contents, the culture vessel containing the growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).
5. Incubate the culture at 37°C in a suitable incubator.  A 100% air atmosphere is recommended if using the medium described on this product sheet. Note: This cell line grows slowly. 

Subcultivation ratio: A subcultivation ratio of 1:2 is recommended.
Medium renewal: every 2 to 3 days
Note: This cell line grows slowly.

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.

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

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Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.