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By Joe W. Gray and Johan S. Ploem (Eds.)

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Production of such cell populations requires culture conditions that maintain high cell viability, rapid cell growth and a high growth fraction. Unfortunately, a universally 41 3. CELL CULTURE FOR CHROMOSOME ISOLATION useful set of culture conditions has not been devised. Thus, careful attention must be given to optimizing the culture of each cell type to be flow karyotyped. Such optimization has already been accomplished for a variety of cell types including human fibroblasts, amniocytes, lymphocytes and lymphoblastoid cells.

In fact, it is not uncommon to find that as many as 75% of the objects in a chromosome preparation are nuclei, clumps and small debris particles so that, in actuality, high-speed sorting rates are around 200/sec and conventional sorting rates are around 25/sec. The true chromosome sort rate can be increased by raising the lower-level discriminator in the sorter so that the system does not "see" the small debris particles, thereby raising / ยท . However, this comes at the expense of reduced purity since the small debris fragments are no longer rejected by the anticoincidence circuitry.

Continued work will be required, however, to establish cell culture conditions that allow routine application of flow karyotyping to solid tumors. 5. 6. 7. 8. 9. ACKNOWLEDGEMENTS I wish to thank Kerry Brookman for pointing out the needs of other technologists and Richard Langlois and Irene Jones for many helpful comments. I thank Joe Gray for final editing of this chapter. This work was performed under the auspices of the US Department of Energy by the Lawrence Livermore National Laboratory under contract number W-7405-ENG-48 with support from USPHS grant 17665.

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