BRIEF REPORT |
DAPI Fluorescence in Nuclei Isolated from Tumors
Division of Experimental Therapeutics, Radiation Oncology Department, Sylvester Cancer Center, University of Miami, School of Medicine, Miami, Florida
Correspondence to: Dr. Awtar Krishan, Division of Experimental Therapeutics (R-71), University of Miami, School of Medicine, PO Box 01690, Miami, FL 33136. E-mail: akrishan{at}med.miami.edu
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Summary |
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(J Histochem Cytochem 53:10331036, 2005)
Key Words: tumors nuclear volume flow cytometry DAPI DNA content
4,6-DIAMIDINO-2-PHENYLINDOLE DIHYDROCHLORIDE (DAPI), a DNA-binding fluorochrome, is extensively used for the determination of nuclear DNA content and for cell cycle analysis (Kapuscinski 1995). In live cells, DAPI fluorescence is slow to appear and often necessitates long staining times (Otto 1994
), but in isolated nuclei, Darzynkiewicz et al. (1984)
reported that staining equilibrium was achieved in approximately 5 min.
During the course of an investigation on human breast tumors, we were intrigued by some samples that failed to yield high-resolution DNA histograms after staining with nuclear isolation medium4,6-diamidino-2-phenylindole dihydrochloride (NIM-DAPI) (Wen et al. 2001), while aliquots of the same tumor stained with propidium iodide/hypotonic citrate (Krishan 1975
) yielded reasonably good histograms. In most of the tumors with a broad CV of the G0/G1 peak, the nuclear volume vs DNA content plots also had a prominent slope. Because none of the earlier reports had dealt with this artifact, the present study was undertaken to determine the reason for the broad CV of the DNA histograms and slope of the volume vs DNA content of tumors stained with NIM-DAPI.
Breast tumor biopsies were obtained from the NIH-sponsored Cooperative Human Tissue Network at the University of Alabama, Birmingham, and stored at 80C. A small piece of the frozen breast tumor was minced in PBS using surgical forceps and a scalpel. The suspension was aliquoted into tubes containing three different concentrations of DAPI [NIM-DAPI (Wen et al. 2001) and a 1:2 dilution of NIM-DAPI with NIM or PBS] and our DAPI formulation (3 µg/ml DAPI containing 0.1% NP40 in PBS). Samples (at an approximate concentration of 106 nuclei/ml) were vigorously pipetted and filtered through 40-µm nylon cloth before analysis.
Initial studies were performed on a NASA/American Cancer Society flow cytometer fitted with a mercury lamp and means for simultaneous measurement of electronic volume and DNA-DAPI fluorescence through a solid-state photodiode (Thomas et al. 2001). In subsequent studies, we used an NPE Quanta flow cytometer (a commercial and advanced version of the NASA/American Cancer Society flow cytometer manufactured by NPE Systems, Pembroke Pines, FL) in which the photodiode was replaced with two photo-multiplier tubes, dichroic mirror, and band pass filters for collection of the blue (450 nm) and red (580 nm) fluorescence. ModFit program (Verity Software House, Inc.; Topsham, ME) was used for cell cycle analysis. WinMDI 2.8 software (©19931998 Joseph Trotter, downloaded from http://facs.scripps.edu) was used for data analysis and graphics.
Figure 1 shows DNA histograms (Figures 1A1C) and nuclear volume vs DNA content dot plots (Figures 1D1F) of nuclei from a breast tumor and trout red blood cells stained with NIM-DAPI and analyzed on the NASA/American Cancer society flow cytometer fitted with a photodiode detector. The DNA histogram (Figure 1A) shows a broad CV, and the dot plot of DNA vs nuclear volume (Figure 1D) has a prominent slope. Histogram 1B and dot plot 1E are of the sample shown in Figure 1A after dilution with 1:3 parts of PBS. The slope of the DNA vs nuclear volume (Figure 1E) was reduced in this sample. Figures 1C and 1F show nuclei stained with NIM-DAPI and analyzed after centrifugation and resuspension in PBS. The CV of this DNA distribution had significantly improved, and the slope seen in Figure 1D was not evident in the DNA vs nuclear volume plots (1F). These observations would suggest that the broad CV and the slope of the DNA vs volume plots seen in nuclei stained with NIM-DAPI are related to the high DAPI concentration and the resulting nonspecific binding.
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In an earlier review, Kapuscinski (1995) reported that DAPI was first used for the isolation of mitochondrial DNA in cesium gradients, and that its fluorescence was enhanced on binding to adenine and thymine (AT)-rich DNA. Because RNase digestion had no effect on DAPI fluorescence, it was assumed that binding was specific for the double-stranded DNA. Subsequent studies have confirmed binding of DAPI in the AT-rich regions of the DNA minor groove. DAPI is a fluorochrome of choice for monitoring of mycoplasma contamination, measuring low amounts of DNA in cellular homogenates and for flow cytometric evaluation of DNA content (Kapuscinski 1995
). The 340-nm absorption maxima of DAPI is suitable for excitation from a mercury source (filtered through a UG 1 filter) or UV laser lines of 351 and 364 nm. On binding to DNA, there is nearly a 20-fold increase in DAPI fluorescence, and removal of histones by 0.1 N HCL will nearly double this fluorescence (Darzynkiewicz et al. 1984
).
Besides binding in the minor grove of AT-rich DNA sequences, DAPI can bind to other cellular components. At high concentrations, DAPI can precipitate and condense double-stranded nucleic acids. The DAPI RNA complex has an emission maximum of 500 nm as compared with 448 nm emission with double-stranded DNA. However, fluorescence of the DAPIRNA complex is 20% that of the DAPI bound to DNA. Kapuscinski (1990) and Tijssen et al. (1982)
have mentioned that DAPI can form higher wavelength-emitting complexes with cellular components other than DNA. DAPI can also bind to tubulin, although fluorescence of the resulting complex is less than that of the DAPIDNA complex.
From data shown in the present report, it is clear that the broad CVs seen in some of the nuclei stained with higher DAPI concentrations were due to the red emission of DAPI. Thus for generation of high-resolution DNA histograms, one can either collect only the blue fluorescence emission, exclude the red emission by use of barrier filters, or use a lower concentration of DAPI. As pointed out by Taylor and Milthorpe (1980) and in our earlier publication (Krishan et al. 2001
), the proper concentration of DAPI needed for generation of high-resolution DNA histograms differs from cell line to cell line and may need to be individually determined for a particular specimen.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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