The paper by Dominika Zurek-Biesiada and coworkers in the current issue of Cytometry A (page 441) focuses on three important DNA binding fluorochromes, DAPI or 4′,6-diamidino-2-phenylindole, Hoechst 33258, and DCV or Vybrant® DyeCycle™ Violet. These fluorochromes are extensively used in laser flow and image cytometry for estimation of DNA content and for cell cycle analysis (1–3).
DAPI is extensively used for high resolution DNA analysis using UV excitation from a high pressure mercury lamp (2, 3). On binding to DNA, DAPI emits blue fluorescence. Results (spectral emission and resolution of DNA distribution peaks) in cells stained with DAPI are pH- and concentration-dependant and one needs to adjust the dye concentration and pH to get optimum results (4). In some of the earliest papers on DNA flow cytometry, Ghöde and coworkers used DAPI and the Phywe Impulse Flow Cytometer to produce some of the best high resolution DNA distribution histograms of tumor cells and X and Y bearing sperms (2, 3).
The fluorochrome Hoechst 33342 which is similar to Hoechst 33258 (1) was used as a vital DNA binding stain and shown to be a substrate for the efflux pump (5). In subsequent studies, Hoechst 33342 fluorochrome was used to identify the side-population (SP) stem cells which can be identified on the basis of their reduced drug retention (due to drug efflux) in red vs. blue emission two color dot plots (6, 7). As the Hoechst 33342 needs a UV source of illumination for detection of SP cells, Telford et al. (8) reported that the DNA binding fluorochrome DCV or Vybrant® DyeCycle Violet could be used instead of the Hoechst 33342 by using excitation from a violet laser for the detection of SP cells. Recently, Boesch et al. (9) have shown that DCV is a substrate for the P-glycoprotein pump.
Observations in the present paper by Zurek-Biesiada and coworkers suggest that these three dyes on excitation by a UV beam are protonated and the photo-converted end product can be excited by the blue emission from a 488 nm laser line and will emit green-orange fluorescence. This photo-conversion is reversible and independent of dye concentration or the need for DNA binding. Although this may not be a problem for use of these fluorochromes in routine DNA flow cytometric analysis, in image cytometry due to extended exposure to the UV light this may lead to artifacts. This concern becomes of importance in experiments where a UV beam is used for excitation of the DAPI bound to DNA and blue excitation from a 488 nm laser line may be used for monitoring of markers labeled with other blue excitable fluorochromes. Thus caution may be needed in interpretation of data from polychromatic cytometric analysis where UV excitation is used for analyzing UV excited blue emission and excitation from a blue laser is used to monitor emission of green, yellow, or orange fluorescence.