DNA damage assessment in the adult Drosophila brain via comet assay

Prepare a Lysis solution for one slide by mixing 20mL of lysis buffer (Trevigen Comet Assay Reagent Kit) and 2mL of DMSO

Melt LMAgarose in a boiling water bath, aliquot to 1.5ml tubes, and place at 37°Cuntil use

Place comet slide at 37°Cuntil use

Dissect 2 adult fly brains per the desired genotype in ice-cold PBS

Homogenize brains with a blue pestle

Combine head homogenate with 100µL 37°C agarose and immediately pipette 75µLonto Comet Slide

Place slides flat at 4 °C for 20 minutes

Immerse slides in prechilled Lysis Solution On ice for 0h 45m 0s

 Immerse in a freshly prepared alkaline solution (prepare 25mLl by mixing 0.3g NaOH and 125µL of 200mM EDTA in dH2O). pH>13 for 0h 30m 0s at Room temperature

Drain excess buffer and wash in 1X TAE twice for 0h 5m 0s each

Run for 0h 10m 0s at 23V/6mA in TAE_one volt per cm electrode to the electrode

Drain excess buffer and rinse in dH₂O

Immerse slides in 70% Ethanol for 0h 5m 0s and then air-dry slides 0h 5m 0s

The next day, prepare SYBR Green (Thermofisher) dilutant by mixing 1µL in 10mL of TE buffer (10 mM Tris-HCL pH 7.5, 1 mM EDTA in dH₂O) and place 50µL on each circle of the comet assay slide for 0h 5m 0s

Drain excess buffer and mount slides without DAPI in permount (Fisher Scientific)

After mounting and drying the slides, take images with 20X objective of the epifluorescence microscope and analyze the comet tails using the Image J Comet assay plugin