An improved acriflavine-Feulgen reagent for quantitative DNA cytofluorometry

Area of Science:

• Histochemistry
• Molecular Biology
• Cytometry

Background:

• The acriflavine-Feulgen method is used for DNA quantification.
• Conventional protocols can result in significant background staining and lower fluorescent intensity.

Purpose of the Study:

• To modify the acriflavine-Feulgen histochemical method for improved quantitative DNA determination.
• To enhance fluorescent intensity and specificity of DNA staining in cells.

Main Methods:

• Modified the acriflavine-Feulgen staining by dissolving acriflavine in 90% ethanol.
• Performed quantitative determination of DNA on hydrolyzed cells affixed to cover slips.
• Analyzed fluorescence spectra and intensity of stained nuclei.

Main Results:

• The modified method significantly decreased non-Feulgen (background) staining in nuclei and cytoplasm.
• Increased fluorescent intensity of Feulgen-stained nuclei by over four-fold compared to conventional methods.
• Achieved a fluorescence maximum of 507 nm, consistent with acriflavine bound to apurinic acid.

Conclusions:

• The modified acriflavine-Feulgen method provides superior staining specificity and quantitative accuracy for DNA measurement.
• This improved method is more satisfactory for in situ DNA quantification by cytofluorometry than conventional protocols.