Chromosome Measurement andSorting byFlowSystems (isolated chromosomes/purified chromosomes/flow microfluorometer/DNA cytophotometry/karyotype)

Aflowmicrofluorometer wasusedtomea- suremetaphase chromosomes insuspension atratesupto 100,000 permin.Chromosomesfromcells oftheChinese hamsterM3-1cell linewereisolated, stained forDNA with thefluorescent dyeethidiumbromide, andanalyzed for DNA content. Ninedistinct peakswereresolved thatcor- respondwellwithindependent chromosomal DNA mea- surementsmade witha high-resolution scanning cyto- photometer. Chromosomesweresortedfromeachpeak byan electronic cellsorter. Visualexamination ofeach fraction indicated thepurityofthesorted chromosomes. Thisnoveltechnology allows separation ofpurified popula- tionsofindividual chromosomessuitable forbiochemical andbiological characterizations. Metaphase chromosomes canbedescribed byseveral proper- ties, including length, area, DNA content, andsedimentation velocity (1,2).Ofthese, DNA content isthemostfunda- mental chromosomal descriptor andtheonly oneinsensitive to shape anddegree ofcompaction. Heretofore, chromosomal DNA content hasbeenmeasured cytophotometrically incon- ventional slide preparations (2-4); themethod iscomplex and time-consuming, andcannot beusedtoprepare fractions of purified chromosomes. Bulktechniques suchascentrifugation cansort thechromosomes, butonly into gross fractions oflow purity (5). Theadvent offlowmicrofluorometry andflow sorters allows application ofprecise DNA measurement, rapid processing, andindividual sorting tometaphase chromosomes. Wereport herethefirst suchapplication. Individual chromo- somesofaclonal derivative (650A) fromtheM3-1Chinese hamster cell line wereisolated, stained forDNA withethid- iumbromide, andanalyzed bytheflowsystem. A distribu- tion ofDNA forthechromosomes insuspension wasobtained thatshowedninedistinct peaks. Thechromosomes corre- sponding toeachpeakwereseparated forvisual scoring byan electronic cell sorter. Therelative DNA content (peak mean), chromosomal frequency (peak area), andchromosome type (peak content asdetermined visually) werecompared with measurements ofsimilar parameters madewithahigh-resolu- tionscanning cytophotometer onchromosomes frommeta- phase spreads ofcells fromthesameculture. Theagreement wasexcellent.