FB2

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.

- The cells are supplied in two different types of glass ampules. One is a

standard ampule, the neck of which must be scored with a sharp file that has

been immersed in ethanol. A definitive sharp nick about 1/8" in length on one

side is necessary. The second type is prescored and is identifiable by a gold

band around the ampule neck, and should not be scored with a file.

- Break the neck of the ampule between several folds of a sterile towel.

- 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 CO₂ will be required.

- It is not necessary to remove the freezing additive. However, if desired, the

culture medium may be changed to remove the protective freezing additive

(dimethylsulfoxide) 24 hours after thawing. If it is desired that the freezing

additive be removed immediately, or that a more concentrated cell suspension be

obtained, centrifuge the above diluted suspension at approximately 125 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

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 10(5) viable cells/ml. Maintain cultures at cell

concentrations between 5 x 10(4) and 2 x 10(5) viable cells/ml.

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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 300 x g for 15 minutes. Resuspend the cell pellet in 10-12 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 5-8 x

10(4) viable cells/ml. If the suspension needs to be diluted use the shipping

medium. Incubate the culture in a flat position at 37°C in a 5% CO₂ in air

atmosphere. Maintain the cell density of the culture as suggested under the

subculture procedure described above.

_______________________________________________________________________________

CATALOGUE DESCRIPTION

The hybridoma cell line FB2 secretes a mouse monoclonal antibody (IgG3) that

reacts with phosphotyrosine residues. The line was produced by fusing FOX-NY

myeloma cells with spleen cells from RBF/DNJ. The antibody reacts with several

phosphorylated proteins on Western blots and in immunoprecipitation reactions.

Reference: J. Biol. Chem. 259: 7909-7915, 1984. Submitted by: J. Burkhart and

L.T. Williams, Howard Hughes Medical Institute, University of California, San

Francisco, CA.

                                                                        (2/99)

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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