NIT-2

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.

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 growth medium and spin at approximately 125 x g for 5 to 7 minutes. Discard supernatant.
4. Resuspend the cell pellet with the recommended complete growth medium (see the specific batch information for the culture recommended dilution ratio) and dispense into a 25 cm² or a 75 cm² culture flask. It is important to avoid excessive alkalinity of the medium during recovery of the cells. It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).
5. Incubate the culture at 37°C in a suitable incubator. A 5% CO₂ in air atmosphere is recommended if using the medium described on this product sheet.

1. Remove and discard culture medium.
2. Subcultures are prepared using a cell dissociation buffer (an enzyme‑free Hanks' based solution; GIBCO, Catalog #13150-016). Add 2 mL cell dissociation buffer per 25 cm. sq. flask (5 mL per 75 cm. sq. flask) and gently rock flask to bathe the cells at room temperature for 1 to 2 minutes.
3. Aspirate the solution and discard.
4. Allow the flask to remain at room temperature for 5 additional minutes (total time from initial addition of cell dissociation buffer approximately 7 minutes).
5. FirmLy tap the flask against palm of hand to dislodge cells.
6. Add 5 mL of fresh medium per 25 cm² flask (10 mL per 75 cm²  flask) and triturate up and down directing the stream along the bottom of the flask to dislodge the cells and break up some of the clumps.
7. Add appropriate aliquots of cell suspension to new culture vessels. 
8. Place culture vessels in incubators at 37°C.

Subcultivation Ratio: 1:2 to 1:3
Medium Renewal: Twice weekly

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.  

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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