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Related Experiment Videos

DAPI fluorescence in nuclei isolated from tumors.

Awtar Krishan1, Payal D Dandekar

  • 1Division of Experimental Therapeutics, Radiation Oncology Department, Sylvester Cancer Center, University of Miami, School of Medicine, Miami, FL 33136, USA. akrishan@med.miami.edu

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society
|August 2, 2005
PubMed
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Broad DNA histogram peaks in human solid tumors are often caused by red fluorescence from NIM-DAPI staining. Diluting or resuspending nuclei in PBS reduces this red fluorescence, improving DNA content analysis.

Area of Science:

  • Oncology
  • Molecular Biology
  • Biotechnology

Background:

  • DNA histograms are crucial for analyzing cell cycle and ploidy in human solid tumors.
  • Broad G0/G1 peaks and prominent slopes in nuclear volume vs DNA scattergrams can hinder accurate analysis.
  • Nuclear isolation medium--4,6-diamidino-2-phenylindole dihydrochloride (NIM-DAPI) is used for DNA staining.

Purpose of the Study:

  • To investigate the cause of broad G0/G1 peaks and prominent slopes in DNA histograms of human solid tumors.
  • To identify factors contributing to variability in DNA content analysis using NIM-DAPI staining.

Main Methods:

  • Nuclei were isolated from frozen human breast tumors.
  • Nuclei were stained with NIM-DAPI.
  • Analysis was performed on stained nuclei, including two-color (blue vs red) fluorescence analysis.

Related Experiment Videos

  • Nuclei suspensions were analyzed after dilution or resuspension in phosphate-buffered saline (PBS).

    • Main Results:

    • Broad G0/G1 peaks and prominent slopes were observed in DNA histograms and scattergrams.
    • Two-color analysis revealed that red fluorescence from NIM-DAPI staining was a major contributor to the observed broadness and slope.
    • Dilution or resuspension of nuclei in PBS significantly reduced the red fluorescence.
    • This reduction in red fluorescence led to the elimination of the slope and a tighter CV of the G0/G1 peak.

    Conclusions:

    • The red fluorescence of NIM-DAPI is a significant factor causing broad DNA histogram peaks and slopes in human solid tumors.
    • Optimizing staining and analysis conditions, such as by diluting or resuspending nuclei in PBS, can improve the accuracy of DNA content analysis.
    • These findings are important for reliable tumor ploidy and cell cycle assessment in cancer research.