Evaluation of a Feulgen type reaction in suspension using flow microfluorimetry and a cell separation technique

The major problem in the application of flow microfluorimetry for quantitation of the total amount of a substance in individual cells is the elimination of cell clumping. Despite the laminar flow conditions of the sample stream carrying the cells through the optical system, elimination of cell clumping without any additional manipulations seems practically impossible. The following steps were shown to be important in almost eliminating cell clumping without using methods which may destroy cells: low initial total number of cells; fixation with 50% methanol for 3 to 12 hr; resuspension of the cell pellet before (in the 0.5 cc left after aspiration of reagent) and after the addition of the next reagent with a pipette, and the use of an optimum centrifugation speed, which in this work did not exceed 500 g. The most essential problem in the use of a fluorescence reaction for quantitative purposes is the proportionality between fluorescence emission and amount of substance to be quantitated, in this case, DNA. Several systems have been used in the past for this purpose, the most common being the nuclei of mouse liver cells with different ploidy levels. In this study, in addition to the linear relation between ploidy levels and fluorescence intensities a cell separation technique was used in an attempt to obtain pure populations of cells in the different phases of the cell cycle. One would expect to observe doubling of the DNA content with increasing cell size and hence fraction number as cells progress from G ₁ through S to G ₂. With the quantitative fluorescence staining method reported, the fluorescence intensities are shown to correspond to the DNA content of single cells. A cell separation technique was evaluated and it was shown that it can be used to obtain cell populations enriched in pre and post mitotic cells or cells in the DNA synthesis phase. Preliminary results have indicated that the staining technique will be extremely useful in the evaluation of the effects of the different cytotoxic drugs on cell cycle kinetics of cells grown asynchronously in logarithmic phase of growth. Such cell kill kinetics will be reported later.