SCGE assay (comet assay)

After sacrifice of the mice, samples of the liver, brain, testis and bone marrow were extracted for the following experiments. Cells were diluted to about 1 × 10⁶ cells/mL with PBS and refrigerated at 4 °C. One hundred microliters of normal-melting agarose were placed on single surface grinding board at 4 °C for 40 min to get cooled and curdled. The cell suspension (50 µL) and low-melting agarose (50 µL) were mixed and spread on glass slides for 40 min at 4 °C. Finally, the sides were covered with low-melting agarose (100 µL) for 20 min at 4 °C. The slides were immersed in a pre-prepared lysing solution at darkness at 4 °C for 1 h. The slides were next immersed in a cold alkaline solution (pH > 13) for 30 min to precipitate DNA. Thereafter, electrophoresis (25 V, 300 mA) was performed for 30 min. The cells were neutralized by neutralizing fluid (0.4 M Tris, pH 7.5), and stained by using 10 µg/mL ethidium bromide. The comet's image was captured by a reversed fluorescence microscope (OLYMPUS IX71). Next, fifty cells from each of three independent experiments were analyzed with Comet Assay Software Project (CASP) 1.2.2. The tail length (measured from the right edge of the comet head), tail DNA percentage and tail moment (tail length × tail DNA percentage) represent the degree of DNA damage¹⁴.