Jan 29, 2024

Public workspaceDNA damage assessment in the adult Drosophila brain via comet assay

DOI
dx.doi.org/10.17504/protocols.io.eq2lyjrrplx9/v1
  • 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
Beatrice Weykopf
Open access
DOI: dx.doi.org/10.17504/protocols.io.eq2lyjrrplx9/v1
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
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 Amount20 mL of lysis buffer (Trevigen Comet Assay Reagent Kit) and Amount2 mL of DMSO

Melt LMAgarose in a boiling water bath, aliquot to 1.5ml tubes, and place at Temperature37 °C until use

Place comet slide at Temperature37 °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 Amount100 µL Temperature37 °C agarose and immediately pipette Amount75 µL onto Comet Slide

Place slides flat at 4 °C for 20 minutes
Immerse slides in prechilled Lysis Solution TemperatureOn ice for Duration00:45:00

45m
 Immerse in a freshly prepared alkaline solution (prepare Amount25 mL l by mixing Amount0.3 g NaOH and Amount125 µL of 200mM EDTA in dH2O). pH>13 for Duration00:30:00 at TemperatureRoom temperature in the dark

30m
Drain excess buffer and wash in 1X TAE twice for Duration00:05:00 each

5m
Run for Duration00:10:00 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 Duration00:05:00 and then air-dry slides DurationOvernight

10m
The next day, prepare SYBR Green (Thermofisher) dilutant by mixing Amount1 µL in Amount10 mL of TE buffer (10 mM Tris-HCL pH 7.5, 1 mM EDTA in dH2O) and place Amount50 µL on each circle of the comet assay slide for Duration00:05:00 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