D556

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 Dulbecco's Modified Eagle's Medium (ATCC 30-2002). To make the complete growth medium, add the following components to the base medium:

• Fetal bovine serum (FBS; ATCC 30-2020) to a final concentration of 10%
• L-Glutamine (ATCC 30-2214) to a final concentration of 2 mM

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. Transfer the vial contents to a centrifuge tube containing 9.0 mL complete culture medium and spin at approximately 125 to 400 x g for 8 to 12 minutes.
4. Carefully aspirate the supernatant and discard, leaving the cell pellet.
5. Gently resuspend the cell pellet in 15 mL fresh pre-warmed complete growth medium, and transfer cell suspension into a vented, T75 flask.
6. Place the flask in a 37°C incubator with 5% CO₂ .

Re-seed cells at log phase every 2-5 days when there are numerous, healthy-appearing clusters present in suspension. Pre-warm fresh growth medium prior to use. Swirl the flask gently to evenly distribute cells in medium.

Cultures can be maintained by addition or replacement of fresh medium. Do not over dilute cell culture, it is recommended to add equal volumes of fresh media and perform full changes via centrifugation if media appears acidic.

1. For acidic cultures, centrifuge cells for 5-12 min at 170 to 400 x g.
2. Discard spent media and re-suspend cell pellet in pre-warmed fresh complete growth media.
3. Pipette cells gently to break aggregates.
4. Add the cell suspension to a vented flask.
5. Place the flask in a 37°C incubator with 5% CO2.

Note: Due to large, tight clusters formed by these cells, it may be difficult to accurately measure cell number and viability. Cultures can be maintained by the addition of fresh medium when there are numerous, healthy-appearing clusters present in suspension and pH of medium has decreased. Alternatively, cultures can be established by centrifugation with subsequent resuspension in fresh medium.

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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