MYC CT 9-B7.3

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 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

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 10(5) viable cells/ml. Maintain cell density between

10(5) and 10(6) 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 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 in a 5% CO₂ in air

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

subculture procedure described above.

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NOTE

The MYC CT9-B7.3 hybridoma cells were deposited at ATCC's request by J.M.

Bishop. The cells are commercially valuable and Dr. Bishop has requested that:

1.) In all papers reporting any use of this line a direct reference will be

made to G.I. Evan et al., Mol. Cell. Biol. 5: 3610-3616, 1985.

2.) Any proposed commercial use of the cells should be negotiated with the

Hooper Foundation/NSW 1542, University of California, San Francisco, CA 94143.

3.) No distribution of any of the cells derived therefrom should be made to

third parties.

_______________________________________________________________________________

CATALOG DESCRIPTION

This hybridoma secretes a murine monoclonal antibody (IgG1) that recognizes

human c-myc protein by immunoprecipitation analysis. The line was produced by

fusing Sp2 myeloma cells with spleen cells from a BALB/c female mouse immunized

with synthetic peptide immunogens whose sequences are derived from that of the

human c-myc gene product. Reference: Mol. Cell. Biol. 5: 3610-3616, 1985.

Submitted by: J.M. Bishop and G.I. Evan, University of California, San

Francisco, CA.

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