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

Abstract

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. Key terms: Hoechst 33342, DNA binding, fluorescence spectrumDuring a series of experiments in which we used Hoechst-33342 (Ho-33342) as a vital DNA stain for mouse bone marrow, we encountered a number of anomalies. These were not only dependent on the dyeDNA concentration ratio and staining time (which were to be expected) but also on the wavelength at which the emission spectrum was analysed. In an attempt to produce an internal biological standard, we repeated the staining procedures with chicken thymocytes (CTHY) and discovered similar anomalies in the "DNA" histograms.The Ho-33342DNA emission spectrum is wide and covers a range from about 400 to 600 nm. Our custombuilt flow cytometer, for which preliminary descriptions have been published (12,13) has the capacity to measure fluorescence emission for up to five different wavelength bands simultaneously. During these experiments we noted that the "DNA" histograms obtained with Ho-33342 staining were considerably different when measured on the violet (390-440 nm) and the green (515-560 nm) photomultipliers. This paper describes our experiences with the chicken thymocytes and discusses some possibilities for the phenomena observed.phate-buf€ered saline, PBS (pH 7.21, the cells were resuspended in Iscove's serum-free medium, pH 7.2, at a concentration of 106/ml. StainingA stock solution of Hoechst-33342 (Calbiochem, La Jolla, CA) was dissolved in distilled water at a concentration of 1 mM. Aliquots of cells were then stained by adding either 1 or 5 p1 of the stock Ho-33342 solution to 1 ml of the cell suspensions to give final stain concentrations of either 1 or 5 pM. The stained cells were then incubated at 37°C in the dark. The samples were analysed at 15, 30, 45, and 125 min after addition of the stain. Flow CytometryThe 164-05 argon laser (Spectra-Physics, Mountainview, CA) of our flow cytometer was tuned to the UV lines (351 + 363 nm), and the fluorescence emission spectrum was analysed on two photomultiplier tubes that admitted light through bandpass filters of 390-440 MATERIALS AND METHODS CellsThymocytes were obtained from a 6-wk-old commercial white-leghorn-based hybrid chicken. A single cell suspension was obtained by teasing the thymus and filtering through ...

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