DSCS

The base medium for this cell line is ATCC-formulated DMEM (ATCC 30-2002). To make the complete growth medium, add the following components to the base medium:

• Fetal bovine serum (ATCC 30-2020) to a final concentration of 10%
• L-Glutamine to a final (ATCC 30-2214) concentration of 1mM

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 150 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 T-75 flask.
6. Place the flask in a 37°C incubator with 5% CO₂ .

It is recommended to subculture at less than 90% confluence. Do not allow cultures to become over confluent as cell may differentiate if allowed to become fully confluent.

Volumes used in this protocol are for 75 cm² flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. 

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with PBS to remove all traces of serum that contains trypsin inhibitor.
3. Add 2.0 to 3.0 mL of Trypsin/0.53 mM EDTA (ATCC 30-2101) to flask and observe cells under an inverted microscope until the cell layer is dispersed (usually within 5 minutes).
   Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Centrifuge the cell suspension at approximately 150 to 400 x g for 8 to 12 minutes to remove dissociation agent.
6. Resuspend the cell pellet in an appropriate amount of complete culture medium.
7. Add appropriate aliquots of the cell suspension to culture vessels.
   Cultures can be established between 1.0 x 10⁴ and 2.0 x 10⁴ viable cells/cm².
8. Incubate cultures at 37°C.

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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