Problem of size dependence in fluorescence DNA cytometry

The relationship among fluorescence intensity, area, thickness, and DNA concentration of DNA-bearing polyacrylamide film, stained with 50 ng/ml DAPI, was studied using an epi-illumination cytofluorometer. Measurements and theoretical analysis suggest the inevitability of the size-dependent effect on human cell nuclei in which the order of the DNA concentration is approximately 10 mg/ml, and that this effect can be virtually negated if the nuclei are stretched and flattened to less than 0.33 microns in thickness on the smears. Alternative practical ways to minimize this effect are to decrease the dye concentration of a staining solution and to make smears as thin and uniform as possible by some means such as hypotonic treatment and/or using an automatic centrifuge smearing apparatus. The size-dependent effect, nevertheless, should be taken into account particularly when determining the DNA content of different cell types and the DNA aneuploidy of tumor cells, regardless of the technique used for measurement.