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ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.
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 xg for
10 minutes, discard the fluid and resuspend the cells with growth medium at the
dilution ratio given in the specific batch information above.
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Cathcart MK, Murakami MNonspecific suppression of antibody synthesis by products of human T lymphocyte hybridomas. Use of a newly selected parent fusion lineIn: Cathcart MK, Murakami MT cell hybridomasBoca Raton, FLCRC Presspp. 227-236, 1985
. Human T lymphocyte hybridomas: production of nonspecific suppressor factors of antibody synthesis. Kyoto, Japan: 5th International Congress of Immunology; 1983.
. . Clin. Res. 31: 734A, 1983.