Jan 29, 2024
DNA damage assessment in the adult Drosophila brain via comet assay
- Mel Feany1,2
- 1Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Mel Feany 2024. DNA damage assessment in the adult Drosophila brain via comet assay. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyjrrplx9/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 29, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94347
Keywords: ASAPCRN, dna damage assessment in the adult drosophila brain, dna damage in the adult drosophila brain, dna damage, adult drosophila brain, drosophila, dna, comet, assay
Abstract
This protocol describes how to determine DNA damage in the adult drosophila brain using the comet assay
Prepare a Lysis solution for one slide by mixing 20 mL of lysis buffer (Trevigen Comet Assay Reagent Kit) and 2 mL of DMSO
Melt LMAgarose in a boiling water bath, aliquot to 1.5ml tubes, and place at 37 °C until use
Place comet slide at 37 °C until use
Dissect 2 adult fly brains per the desired genotype in ice-cold PBS
Homogenize brains with a blue pestle
Combine head homogenate with 100 µL 37 °C agarose and immediately pipette 75 µL onto Comet Slide
Place slides flat at 4 °C for 20 minutes
Immerse slides in prechilled Lysis Solution On ice for 00:45:00
45m
Immerse in a freshly prepared alkaline solution (prepare 25 mL l by mixing 0.3 g NaOH and 125 µL of 200mM EDTA in dH2O). pH>13 for 00:30:00 at Room temperature in the dark
30m
Drain excess buffer and wash in 1X TAE twice for 00:05:00 each
5m
Run for 00:10:00 at 23V/6mA in TAE_one volt per cm electrode to the electrode
10m
Drain excess buffer and rinse in dH2O
Immerse slides in 70% Ethanol for 00:05:00 and then air-dry slides Overnight
10m
The next day, prepare SYBR Green (Thermofisher) dilutant by mixing 1 µL in 10 mL of TE buffer (10 mM Tris-HCL pH 7.5, 1 mM EDTA in dH2O) and place 50 µL on each circle of the comet assay slide for 00:05:00 in the dark
5m
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