VMM5A

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.

Thaw ampoule in 37^oC water bath for approximately 2 minutes.  Transfer thawed cell suspension to a 15.0 mL centrifuge tube containing 9 mL complete medium.  Mix suspension by gentle inversion.  Remove 0.5-1.0 mL for cell count.  Centrifuge remaining suspension in the 15 mL centrifuge tube at 175-195 x g (1000 rpm in an IEC HN SII centrifuge or equivalent) for 5 minutes, RT˚.  Discard supernatant and gently resuspended pellet in 5ml fresh complete medium.  Transfer 5 ml re-suspended cells into 1 T-75 flask containing 10 ml fresh medium. Place the cells in a 5% CO₂ incubator @ 37^oC

Volumes are for a T-75 flask; Adjust accordingly

1. Remove and discard the cell culture medium from the flask.
2. Rinse the cell monolayer with Dulbecco’s PBS without calcium or magnesium and remove.
3. Add 3 to 4 ml of the trypsin-EDTA solution, rotate flask to rinse cell monolayer, remove trypsin solution, and incubate at 37^oC.
4. Once the cells appear to be detached, add 10 ml of complete growth medium with a pipette to the cell suspension to inactivate the trypsin. Gently wash any remaining cells from the growth surface of the   flask. Check the  cells with the microscope to be sure that most (>95%) are single cells. If cell clusters are apparent, continue to disperse the cells with gentle pipetting.
5. Subculture as necessary.
6. Place the flask back into the incubator. Examine the culture the following day to ensure the cells have reattached and are actively growing.
7. Repeat when cells reach confluence.

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 If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.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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