BW5147.G.1.4.OUAR.1

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.

HANDLING PROCEDURE FOR FROZEN CELLS

- Initiate culture as soon as possible upon receipt.

- Thaw by rapid agitation in 37°C water bath. Thawing should be rapid (within

40-60 seconds). As soon as the ice is melted, remove the ampule from the water

bath and immerse in 70% ethanol at room temperature. All of the operations from

this point on should be carried out under strict aseptic conditions.

- Transfer the cell suspension and dilute it with the recommended culture

medium in a culture flask (see specific batch information above for dilution

ratio); incubate at 37°C with 5% CO₂ in air atmosphere. Since it is important

to avoid excessive alkalinity of the medium during recovery of the cells, it is

suggested that the culture medium be placed into the culture flask, tube, etc.

and the pH be adjusted, as necessary, prior to the addition of the ampule

contents. Note that the bicarbonate content of the culture medium will

determine whether an atmosphere containing 5% or 10% CO₂ will be required.

-          It is not necessary to remove the freezing additive (dimethylsulfoxide).

-       If it is desired that the freezing additive be removed immediately, centrifuge the above diluted suspension at approximately 200 x g for 10 minutes, discard the fluid and resuspend the cells with growth medium at the dilution ratio given in the specific batch information above.

FLUID RENEWAL

Add fresh medium (depending on cell density) every 2-3 days.

SUBCULTURE PROCEDURE

Cultures can be maintained by the addition of fresh medium or replacement of

medium. Alternatively cultures can be established by centrifugation with

subsequent resuspension at 5 x 10(4) viable cells/ml. A maximum of 1-2 x 10(6)

viable cells/ml is obtainable.

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HANDLING PROCEDURE FOR FLASK CULTURES (SUSPENSION)

The flask was seeded with cells (see specific batch information above for

concentration), grown and completely filled with medium to prevent loss of

cells in transit. Upon receipt incubate the flask in an upright position for

several hours to return the flask contents to 37°C. After the temperature has

equilibrated, aseptically remove the entire contents of the flask and

centrifuge at 200 x g for 10 minutes. Resuspend the cell pellet in 10 ml of

the shipping medium. From this suspension remove a sample for a cell count and

viability so that the cell density of the suspension can be adjusted to 2-3 x

10(5) viable cells/ml. If the suspension needs to be diluted use the shipping

medium. Incubate the culture in a flat position at 37°C. The shipping medium

contains reduced sodium bicarbonate suitable for a 5% CO₂ in air incubator.

DMEM usually contains 3.7 grams of sodium bicarbonate per liter and should be

incubated in a 10% CO₂ in air incubator. Maintain the cell density of the

culture as suggested under the subculture procedure described above.

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CATALOG DESCRIPTION

BW5147.G.1.4.OUAR.1 is a mutant subline of BW5147, an AKR/J mouse lymphoma

maintained at the Jackson Laboratory. Mutant sublines resistant to 1 X 10(-4) M

6-thioguanine and to 1 X 10(-3) M ouabain were established by R. Hyman. The

cell line can be used to produce T cell hybrids. Reference: J. Natl. Cancer

Inst. (Bethesda) 52: 429-436, 1974. Submitted by: R. Hyman, Salk Institute, La

Jolla, CA.

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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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