The alkaline (pH >13) single-cell gel electrophoresis assay was performed by a modified method of Singh et al.
29 The solution, 0.65% normal melting agarose (NMA) and 0.65% low melting agarose (LMA), was prepared in Ca
2+, Mg
2+-free phosphate-buffered saline (PBS). Cells were suspended in LMA, and 75 μL LMA-cell suspension were pipetted onto a frosted glass microscope slide precoated with a 100-μL layer of 0.65% NMA. The third layer of 75 μL of 0.65% LMA was finally added. Then the slides were immersed in freshly prepared ice-cold lysis solution (1%
N-lauroylsarcosine sodium salt, 2.5 M NaCl, 100 mM Na
2 EDTA, 10 mM Tris-HCl, 1% Triton X-100, and 10% DMSO [pH 10]) to lyse the cell proteins and allow DNA unfolding. After at least 1 hour at 4°C in the dark, the slides were covered with fresh buffer (1 mM Na
2 EDTA, 300 mM NaOH [pH >13]) in a horizontal electrophoresis unit. The slides were allowed to sit in this buffer for 20 minutes to allow for DNA unwinding. Then the DNA was electrophoresed at 20 V and 300 mA for 20 minutes. Unwinding and electrophoresis were performed at 4°C. The slides were washed gently to remove alkali and detergent in a neutralization buffer (0.4 M Tris HCl [pH 7.5]) and fixed in methanol for 3 minutes, and then were stained with 50 μL ethidium bromide (20 μg/mL). All the steps described were conducted under yellow light or in the dark to prevent additional DNA damage. Pictures of 300 cells per treatment sample (100 cells/slide; three replicate slides per experiment per SAR group) were taken individually at 400× magnification under a fluorescence microscope (BX51; Olympus, Tokyo, Japan) equipped with a 530-nm excitation filter, a 590-nm emission filter, and a digital camera (DP50; Olympus). Nuclear width and extent of migration of DNA fragments, mean tail length, and mean tail moment were analyzed using the Image-Pro Plus program (Media Cybernetics Inc.).