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Monochromosomal hybrid cell lines containing human chromosomes in a Chinese hamster background can be maintained in a monolayer culture using conventional techniques. Cells subdivide approximately every 5-7 days in DME medium supplemented with 5% fetal bovine serum (FBS), 5% calf serum (CS), proline, glutamine, and antibiotics. Somatic cell hybrids containing human chromosomes and the radiation hybrid cell lines derived from them contain an inserted histidinol dehydrogenase gene which confers the ability to grow in a selective medium containing histidinol instead of histidine ( 1 3) . These cell lines are grown in a similar medium as described above except that the medium lacks histidine and is supplemented with 0.5 mM histidinol. For harvest, the cells are trypsinized 1 8 h prior to the addition of Colcemid, and replated at a density that should be subconfiuent by the time of harvest. Thirty-two microliters of stock Colcemid at 1 0 f.Lg/ml (Gibco Life Technologies, Grand Island, NY) is usually added for approximately 90 min prior to harvesting. Harvesting of cultures is performed according to conventional cytogenetic methods, using 0.075 M KCl as the hypotonic agent, and three parts methanol to one part glacial acetic acid as the fixative. After repeated rinsing and centrifugation, the cell suspensions are dropped onto glass slides, which are either air-dried overnight or flamed briefly.