Ploidy of living clones of human somatic cells determined by mensuration at metaphase.

Abstract

C. A. SPRAGUE, H. HOEHN, and G. M. MARTIN. From the Department of Pathology,University of Washington, Seattle, Washington 98195INTRODUCTIONTetraploid clones have been useful for studies ofsomatic segregation (10), mutation (4), and theregulation of protein synthesis (14, 13, 12) . Withthe availability of various heterochromatin mark-ers, such material is also proving valuable forcytological studies ofinterphase (5). Finally, clonesof cells with various ploidies should serve as veryprecise and convenient standards for flow micro-fluorometric assays of cell DNA (8, 1). We reporthere a rapid and simple technique for the diagnosisof ploidy in clones of somatic cells derived fromhuman skin and amniotic fluid. The methodemploys objective measurements on small samplesof living cells. Other methods, such as karyotypingand chemical assays, take much longer and requiresacrifice of all or part of the cultures.MATERIALS AND METHODSStrain 73-4 was derived from the dermis of a 24-yr oldfemale referred for cytogenetic studies because ofreproductive failure Her karyotype was 46, XX.Cultures were examined at both early passages (about10 cumulative population doublings) and middlepassages (about 20 cumulative population doublings)(11). Strain 71-38 was a fibroblast-like culture derivedfrom the skin of a 30-yr old normal male. StrainAC-16 became available after prenatal diagnosis atthe 17th gestational week of a male fetus. Generalculture conditions were as previously described (11).Media was a modification of the Dulbecco-Vogtformulation (3) with 16% (vol/vol) heat-inactivatedfetal calf serum. Cytochalasin B treatments (2 µg/mlfor 36 h) for induction of tetraploidy (6) were carriedout 6-8 h after dilute plating of 40-60 cells in 4 nil ofmedia in 60-mm plastic Petri dishes. The cultureswere then fed every third day. Between days 10 and 15,consecutive clones were analyzed by a two-step pro-cedure: (a) four to 12 living mitotic cells were photo-graphed at the stage of distinct equatorial alignmentof chromosomes using a Nikon inverted microscope(model MS) with a 20X DLL objective and phasecondenser. Measurements of the lengths of thesemetaphase plates were determined from projectionsof the photographic negatives before knowledge ofcytogenetic analysis. The measurements were made aslinear vectors transversely to the major spindle axesfrom mid-outer chromosome to mid-outer chromo-some. (b) Clones were processed for chromosomalanalysis by an