Optimization of mammalian chromosome suspension preparations employed in a flow cytometric analysis.

Abstract

Metaphase chromosomes from Chinese hamster V-79 cells have been stained with propidium iodide and their DNA content was measured by flow cytometry. The chromosomes in suspension must be stabilized during the preparation to preserve their morphological integrity. In order to obtain high resolution in the flow karyotype, several modifications were made to the method used to prepare chromosome suspensions. The influence of several ions, detergents and detergent mixtures was investigated. Magnesium (3-5 mM) together with sodium (10 mM), Tris (10 mM) and Triton X-100 (0.4%) gave the best results. In contrast, sodium and potassium exerted no effect, while calcium had a negative effect on the preparation as measured with flow cytometry. Compared with a solution of potassium chloride + propidium iodide, chromosomes prepared in Tris buffer resolved better with less background. A histogram quality factor which contains the variables peak height, depth of valley and coefficient of variation, was used to quantitate the histograms.