Sperm DNA Integrity Analysis by Flow Cytometry

The sperm aliquots stained with AO were also analyzed using the flow cytometry technique. Briefly, 0.2 mL of thawed samples with 1 million events was diluted with TNE buffer (0.01 M Tris–HCl, 0.15 M NaCl, and 1 mM EDTA, pH: 7.4)
containing 10% glycerol at a density of 1-2×10⁶ sperm/mL. Then, they were immediately mixed with 0.4 mL of the solution containing 0.1% Triton X-100, 0.15 M NaCl,
and 0.08 N HCl (pH 1.2) at 4 °C. After 30 seconds, they were incubated in 1.2 mL of AO at a 6 μg/mL concentration in a solution containing 0.037 M citric acid, 0.126 M Na₂HPO₄,
0.001 M disodium EDTA, and 0.15 M NaCl (pH 6.0) at 4 °C. These were analyzed using the FACSCalibur^TM flow cytometer. Strong green (FL-1)
and negative red fluorescence (FL-2) depicted normal sperm integrity (excitation wavelength at 488 nm and emission wavelengths at 520 nm for FL1 and 640 nm for FL3).
A sample of acid-treated sperm was used as a positive control. DNAf index (DFI) was calculated using the formula below and expressed as a percentage.
DFI=Mean value of red fluorescence/(mean value of red+green fluorescence).
AO shows green fluorescence in the monomeric state (when it binds to DNA) and red fluorescence in the polymeric state (when it binds to RNA or denatured single-stranded DNA). Larger cells with higher histone and lower protamine content are found in the upper quartile of the dot blot chart. These cell populations show high DNA stainability (HDS) and represent immature cells. ^{17 (link)}