Comet Assay for DNA Damage

Comet Assay for DNA Damage
The comet assay was performed using the CometAssay Kit (Trevigen) with minor modifications.
(1) Cracking. Place a petri dish containing 1 mL of ice-cold solution (1ⅹ phosphate-buffered saline and 20 mM EDTA). Chop 0.2 g of anther sample in the petri dish with a scalpel. A 100-mm nylon mesh filter (Millipore) was used to filter the nuclei mixed. 
(2) Sample loading. 50 μL of the nuclei mixed into 300 μL of 1% (w/v) low-melting agarose (LMA agarose, pre-incubated at 37°C) and pipetted onto slides. After coagulation, immerse the slides in 4°C lysis solution (kit provided) for 1 h. Pay attention to handle all process gently. 
Note: the LMA agarose should be melted at 90–100°C water bath for 5 minutes and placed in a 37°C water bath for at least 20 minutes to cool before use.
(3) Unspin. Drain excess buffer from slides and immerse in freshly prepared Alkaline Unwinding Solution (0.3 N NaOH, 5 mM EDTA) for 40 minutes at 4°C, in the dark. Then wash the slides with 1ⅹ TBE for 5min and repeat 2 times.
(4) Electrophoresis. Take out the slides and slides were electrophoresed at 25 V in 1ⅹ TBE for 30 min and then immersed in 70% (v/v) ethanol for 5 min.
(5) Dyeing and take pictures. After drying at room temperature, the slides were stained with SYBR green (1:10,000 dilution; Bio-Rad) and then examined with a Zeiss ImagerA2 microscope. The CometScore software (http://www.autocomet.com) was used to evaluate the percentage of DNA in each comet tail. Use CometScore software (http://www.autocomet.com) to evaluate the percentage of DNA (T DNA%) in each comet tail. The degree of DNA damage of different samples was calculated according to the method described by Wang and Liu (2006).