NB324K

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

• Fetal bovine serum to a final concentration of 5%
• L-Glutamine 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 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 with the appropriate amount of complete growth medium and transfer cell suspension into a vented T-75.
6. Place the flask in a 37°C incubator with 5% CO₂.

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 0.25% 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: This cell line is resilient to manipulation. Cells may be clumpy upon trypsinization. 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 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 new 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.

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.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

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