Flow cytometric fluorescence emission spectrum analysis of hoechst-33342-stained DNA in chicken thymocytes


  • MRC/CORU serial number P1.083.

  • This work was partially supported by research grants R01 AM28587 awarded to A.N. by the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, NIH, DHHS, and research Career Development Award K04 HL00440 awarded to A.N. by the National Heart, Lung and Blood Institute, NIH, DHHS.


Hoechst-33342-stained chicken thymocytes were analysed simultaneously on two fluorescence wavelength bands (green and violet) in our custom-built flow cytometer, and two major subsets were identified. In one subset (33% of the total) the emission spectrum remained constant with time, with little change in the respective green and violet fluorescence intensities. In the other subset (42% of the total) the green fluorescence increased during staining, resulting in a considerable change in the green-to-violet ratio, due to a change in the “shape” of the fluorescence emission with time. The data indicate that two binding sites, or two types of binding at the same site, exist in DNA for this dye and that these have different binding energies and, consequently, different fluorescence emission properties.