Serum-Free Cell Freezing Medium

1. Harvest the culture to prepare a cell suspension using your standard, cell-specific method.
2. Centrifuge the cells at 125 x g for 5 to 10 minutes.
   1. Do not over-centrifuge cells as this may cause cell damage.
   2. After centrifugation, the cells should form a clean loose pellet.
3. Aspirate medium and (neutralized) dissociation solution (used with adherent cells) and resuspend the cell pellet in 2 to 8 mL fresh, pre-warmed, complete growth medium.
4. Count the cells. Centrifuge the cells again at 125 x g for 5 to 10 minutes.
5. Take the Serum-Free Cell Freezing Medium from storage and swirl to mix. Decontaminate the vial by dipping in or spraying with 70% alcohol.
6. Aspirate the medium and suspend the cell pellet in Serum-Free Cell Freezing Medium at a concentration 3 x 10⁶ to 5 x 10⁶ cells/mL. Aliquot 1 mL of the cell suspension to each labeled cryovial.
7. Freeze the cells gradually at a rate of -1°C/min until the temperature reaches -70°C to -80°C. The cells should not be left at -80°C for more than 24 to 48 hours. Once at -80°C, frozen cryovials should be transferred to the vapor phase of liquid nitrogen for long-term storage.

1. Warm the complete growth medium to 37°C prior to use with the cells.
2. Thaw a vial of cells cryopreserved in Serum-Free Cell Freezing Medium 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 90 seconds).
3. Remove the vial from the water bath before the contents are completely 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.
4. Transfer the vial’s contents plus 5 mL of complete growth medium to a 15 mL centrifuge tube. Use an additional 1 mL of medium to rinse the vial and transfer the liquid to the 15 mL tube. Add 4 mL of complete cell growth medium to bring the total volume to 10 mL.
5. Spin the cells at 125 x g for 5 min. Aspirate the supernatant and resuspend the pellet in 2 mL of complete growth medium.
6. Count the cells; adjust the volume so that the cells are plated at the appropriate seeding density.

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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