Quantitative Flow Cytometric Determination of Anthracycline Content of Rat Bone Marrow Cells¹

Flow cytometry was used to determine the daunomycin content of rat bone marrow cells after incubation in vitro. The spontaneous fluorescence of daunomycin was measured upon excitation with laser light at 488 nm. Forward and perpendicular light scatters of the cells were simultaneously measured to allow identification of granulocytic and lymphocytic subpopulations. A linear relationship was found for a 30-min exposure between the drug concentration (ranging from 0.2 to 3 µg/ml) in the incubation medium and the fluorescence intensity for both lymphocytes and granulocytes. Dead cells contaminating cell suspensions showed several times higher daunomycin fluorescence than did viable cells. In fixed cells, the fluorescence reflects daunomycin bound to DNA, since the fluorescence intensity of daunomycin-treated fixed cells returns to the level of unstained cells after DNase treatment.

Quantitation of the cellular drug concentration was done by exposing cells in vitro to varying doses of [³H]daunomycin. At each drug concentration, the fluorescence intensity of the cells was measured using flow cytometry. Granulocytes and lymphocytes were sorted on the basis of light scatter. The amount of intracellular drug was determined for both cell populations at each drug dose by measuring the radioactivity in 600,000 sorted cells. The concentration per cell was on the order of 10^-18 mol. For both the granulocytic and the lymphocytic subpopulations, a linear relationship was found between drug-related radioactivity and fluorescence intensity.