Physical-chemical characterization of living cells by laser-flow microfluorometry

A rapid method for the laser-flow microfluorometry determination of nucleic-acid content per cell is presented. A frequency distribution of fluorescence is obtained from suspensions of living cells treated with ethidium bromide directly in their own medium (or calcium-magnesium-free Hanks' balanced solution). For a fixed number of cells, a frequency distribution of fluorescence is obtained as a function of the amount of ethidium bromide progressively added to the suspension until staturation. At any ratio of added dye per unit of DNA, histograms generated from cells stained with this method give results similar to those generated after fixation and staining by the Feulgen technique, both in terms of cell-cycle phases and ploidy-level determination. The present technique requires a minimal amount of material, is instantaneous, and is conducted directly on living cells. Furthermore, dye concentration-dependence studies of mean fluorescence per cell allow determination of association constant and binding process (primary and secondary) between the intact cell and ethidium bromide. Cells which have the same amount of DNA but vary in the amount of RNA and/or chromatin conformation (like G0 and G1) can then be distinguished.