Human chromosome isolation from short-term lymphocyte culture for flow cytometry

Flow cytometry is uniquely suited for distinguishing between isolated mammalian chromosomes differing in DNA content^1,2 or DNA-base composition^3,4. Using this technique, the 24 types of human chromosomes can be resolved into 15 groups when stained with Hoechst 33258 (refs 3, 5). When Hoechst 33258 (HO) and chromomycin A3 (CA3) are used, 20 groups of human chromosomes can be distinguished by dual-beam flow cytometry ^3,6, and the relative frequency of chromosomes in each group estimated. Flow measurements provide a precise description of the average chromosome complement of the cell population (a flow karyotype⁷) that is sensitive to chromosomal rearrangements^2,8 and frequency changes; and also enable individual chromosomes to be purified, for example, for gene mapping⁹. Until now, chromosomes have been isolated for flow cytometry only from established fibroblast cultures. However, the maintenance of such cultures is time-consuming and expensive, and there is the risk that chromosomal rearrangements may occur during extended fibroblast culture. Here we describe two procedures for the isolation from short-term lymphocyte cultures of metaphase chromosomes that are morphologically intact, suitable for flow cytometric analysis and purification, and have DNA of high molecular weight.