Jun 16, 2026

Comet Assay Optimized for Coral Cells

DOI
https://dx.doi.org/10.17504/protocols.io.kqdg3r4qeg25/v1
  • 1Arizona State University
Avery J Kruger
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DOI: https://dx.doi.org/10.17504/protocols.io.kqdg3r4qeg25/v1
Protocol Citation: Avery Kruger, Liza Roger 2026. Comet Assay Optimized for Coral Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3r4qeg25/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: June 08, 2026
Last Modified: June 16, 2026
Protocol  Integer ID: 318720
Keywords: dna damage in coral cell, coral cells this protocol, coral cell, comet assay, dna damage, cell gel electrophoresi, evaluation of the comet, using electrophoresi, comet, enzo life science, assay, cell
Abstract
This protocol has been adapted from Enzo Life Sciences (found here) and optimized for coral cells. The assay is designed to visualize and quantify DNA damage in coral cells and breakage by using single-cell gel electrophoresis. The evaluation of the comet “tail” is the metric used to assess DNA damage, and can be used in a multitude of tissue types. Samples are lysed and loaded into an agarose gel, stained with dye, and then analyzed using electrophoresis.
Image Attribution
Created by: Avery Kruger on 4.21.2026
Guidelines
Cell suspension samples need to be prepared before this assay can be run. This assay works best with cells in suspension at a minimum of 2.0 x 106 cells/mL. For coral cell dissociation protocols please click on Coral Dissociation Protocol. At the final step of cell dissociation, resuspend cells in PBS instead of cell culture media. Resuspend cells in ice-cold 1X PBS . The media used for cell culture can reduce the adhesion of the agarose on the Comet Slide so do not use it. This protocol was developed by Enzo Life Sciences using 1X PBS, however based on coral cell biology, future trials should test to optimize resuspending cells in 3X PBS.

Materials
Materials supplied in the Enzo Life Sciences kit:
  1. Lysis Solution (2X 500 mL, 2.5 M NaCl, 100 mM EDTA (pH 10), 10 mM Tris Base, 1% sodium lauryl sarcosinate, 1% Triton X-100).
  2. Comet LMAgarose (15 mL, low melting point agarose in PBS).
  3. Comet slide (25 ct).
  4. EDTA (12.5 mL, 200 mM).
  5. CYGREEN Nucleic Dye (20 µL).

Other materials needed:
  1. 20, 200, 1000 micropipettes
  2. Micropipette tips
  3. Water bath capable of heating up to 37 °C and 95 °C.
  4. Horizontal electrophoresis machine (BioRad Mini-Sub Cell Horizontal Electrophoresis System and PowerPac Basic Power Supply)
  5. Fluorescence microscope with FITC channel (ex:498 nm em: 517 nm)
  6. 1 L graduated cylinder
  7. 4 °C fridge/cold room
  8. 10X PBS
  9. NaOH pellets
  10. 0.5 M EDTA (pH 8.0)
  11. 70% ethanol
  12. Alkaline solution (pH >13) (made in Reagent Prep step)
  13. DI water
  14. Gel spatula
  15. Cover slips
Troubleshooting
Problem
Agarose gel
Solution
Sometimes the agarose is thick and hard to load because it has not melted properly. Be sure to melt the agarose at 95C and cool down at 37C to ensure it has gone completely liquid.
Problem
1x PBS
Solution
The original protocol from Enzo uses 1X PBS buffer to resuspend cells. However, since cnidarians prefer higher saline environments, it is recommended that this assay is trialed using 3X PBS to see if there is a better response from the cells in that media than only 1X.
Safety warnings
Work under dim or yellow light to avoid UV damage.
Before start
PPE: Lab coat, gloves, safety glasses, heat-resistant gloves. Reagents with should be prepared fresh.
Reagent Preparation
1x Ca + Mg free PBS
a. Dilute 100 ml 10X PBS with 900 ml DI water to make 1x solution.
b. Store at room temp.
Lysis Solution
a. Chill on ice or in 4C for at least 20 min before use in assay. (Lysis solution is stored in 4C).
Comet LMAgarose
a. Loosen cap and allow the bottle to heat at 95C for 5 min.
b. Place bottle in 37C water bath to cool down for 20 min. The agarose will remain molten at 37C.
c. The solution is ready to use once molten.
Alkaline Solution, pH > 13 ****
a. Add 0.6g NaOH pellets to a beaker.
b. Add 250ul 200mM EDTA
c. Add 49.75ml DI water and mix.
d. Stir together in a beaker in the fume hood. This is an exothermic reaction, so allow the solution to cool completely before storing in 4C.
Alkaline electrophoresis solution pH >13 (300mM NaOH, 1mM EDTA).
a. Prepare a stock solution of 500mM EDTA (pH8).
b. For 1L of solution:
i. Mix 12g NaOH pellets in a beaker
ii. Add 2mL 500mM EDTA (pH8).
iii. Adjust volume to 1L with DI water after NaOH is completely dissolved.
CYGREEN Stock
a. For 10x concentration:
i. Mix 1ul dye with 999ul DI water.
ii. Store in the dark in -20C.
Assay procedure
All steps are performed at room temperature unless otherwise noted. Work under dim or yellow light to avoid UV damage.
Chill lysis solution at 4C for minimum 20 min.
Melt the LMAgarose at 95C for 5 min, then place in 37C water bath for 20 min to cool down. Do not use a heat block.
Combine cells (at 2 x 10^6 cells/mL) with molten agarose at a 1:10 (v/v). Immediately pipette 75ul of sample onto a new Comet Slide.
a. Mix 500ul Comet agarose with 50ul of Cells in 1x PBS; then pull 75ul of mixture.
i. If necessary, use the tip of the pipette to spread the sample over the slide.
ii. When working with many samples it may be convenient to place aliquots of the molten agarose into prewarmed microcentrifuge tubes and place the tubes at 37°C. Add cells to one tube, mix by gently pipetting once or twice, then transfer 75 μL aliquots onto each sample area as required. Then proceed with the next sample of cells.
Use the green gel spatula to quickly scrape agarose from Comet Slide (similar to blood test). This allows the agarose to sit in a uniform flat plane.
Place the slide with the sample flat and in the dark in 4C for 20min.
a. A clear ring (0.5mm) will appear at the edge of the slide area.
b. Increasing gelling time to 30min can improve adherence in high humidity environments.
Then immerse the slide in the pre-chilled lysis solution for 60 min at 4C in the dark.
Tap off excessive buffer from the slide and immerse in the freshly prepared Alkaline solution pH > 13 (Step 4 - Reagent Prep).
Leave the slide in alkaline solution for 45 min - room temp-in the dark.
Transfer the slide from the alkaline solution and place immediately in the horizontal electrophoresis apparatus.
Place the slides flat onto the gel tray and align it so it’s equidistant from the electrodes. Carefully pour Alkaline electrophoresis solution until level and it just covers the sample slides.
Set the voltage to 300mA, and run for 20-40 min at 4C.
Gently tap off excess solution and rinse slides by dipping several times in DI water, and then place in 70% ethanol for 5min.
Air dry the samples.
a. This allows all the cells to move into a single plane for observation.
Place 100ul diluted stain (Step 6 - Reagent Prep) onto each circle of agarose, and stain for 30min-room temp- in the dark.
Gently tap the slide to remove excess stain, and rinse briefly in DI water. Dry slides completely at 37C.
Add a very small drop of water to the corner of the comet slide. Add one cover slip to each well for microscopy.
View slide via FITC channel on the ECHO scope (CYGREEN ex/em: 489/515nm). Image at 20x.
Data analysis
When excited, CYGREEN® Nucleic Acid Dye emits green light. In healthy cells the fluorescence is confined to the nucleoid: undamaged DNA is supercoiled and thus does not migrate very far of the nucleoid under the influence of an electric current. In cells that have accrued damage to the DNA, the alkali treatment unwinds the DNA, releasing fragments that migrate from the cell when subjected to an electric field. The negatively charged DNA migrates toward the anode and the extrusion length reflects increasing relaxation of supercoiling, which is indicative of damage. When using TBE as the electrophoresis buffer, the length of the comet tail may be correlated with DNA damage. When using alkaline electrophoresis conditions, the distribution of DNA between the tail and the head of the comet should be used to evaluate the degree of DNA damage. The characteristics of the comet tail including length, width, and DNA content may also be useful in assessing qualitative differences in the type of DNA damage.
The comet tail can be scored according to DNA content (intensity). The control (untreated cells) should be used to determine the characteristics of data for a healthy cell. Scoring can then be made according to nominal, medium or high intensity tail DNA content. At least 75 cells should be scored per sample.
There are several image analysis systems that are suitable for quantitation of Comet SCGE Assay data. The more sophisticated systems include the microscope, camera and computer analysis package. These systems can be set up to establish the length of DNA migration, image length, nuclear size, and calculate the tail moment. At least 75 randomly selected cells should be analyzed per sample.
Protocol references
Data analysis

  1. Hong, Y., Han, H. J., Lee, H., Lee, D., Ko, J., Hong, Z. Y., ... & Sohn, I. (2020). Deep learning method for comet segmentation and comet assay image analysis. Scientific Reports, 10(1), 18915.
2. Møller, P., Azqueta, A., Sanz-Serrano, J., Bakuradze, T., Richling, E., Eyluel Bankoglu, E., ... & Collins, A. (2023). Visual comet scoring revisited: a guide to scoring comet assay slides and obtaining reliable results. Mutagenesis, 38(5), 253-263.
3. Gyori, B. M., Venkatachalam, G., Thiagarajan, P. S., Hsu, D., & Clement, M. V. (2014). OpenComet: an automated tool for comet assay image analysis. Redox biology, 2, 457-465.
4. Barbé, L., Lam, S., Holub, A., Faghihmonzavi, Z., Deng, M., Iyer, R., & Finkbeiner, S. (2023). AutoComet: a fully automated algorithm to quickly and accurately analyze comet assays. Redox Biology, 62, 102680.
5. Beleon, A., Pignatta, S., Arienti, C., Carbonaro, A., Horvath, P., Martinelli, G., ... & Piccinini, F. (2022). CometAnalyser: A user-friendly, open-source deep-learning microscopy tool for quantitative comet assay analysis. Computational and Structural Biotechnology Journal, 20, 4122-4130.
6. Vojnovic, B., Barber, P. R., Johnston, P., Gregory, H. C., Marples, B., Joiner, M. C., & Locke, R. J. (2013). A High Sensitivity, High Throughput, Automated Single-Cell Gel Electrophoresis (‘Comet’) DNA Damage Assay. Phys. Med. Biol, 58(1), 15.

Assay references

1. Lemay, M. and K.A. Wood, 1999. Detection of DNA damage and identificationof UV-induced photoproducts using the CometAssay™ kit. BioTechniques27(4):846-851.
2. Angelis, K.J., M. Dusinska and A.R. Collins. 1999. Single cell gel electrophoresis: Detection of DNA damage at different levels of sen-sitivity. Electrophoresis 20:2133-2138.
3. Morris, E.J., J.C. Dreixler, K-Y. Cheng, P.M. Wilson, R.M. Gin and H.M. Geller.1999. Optimization of single-cell gel electrophoresis (SCGE) for quantitative analysis of neuronal DNA damage. BioTech-niques 26:282-289.
4. Malyapa, R.S., C. Bi, E.W. Ahern, and J.L. Roti Roti, 1998. Detection of DNA damage by the alkali comet assay after exposure to low dose gamma radiation. Radiation Res 149:396-400.
5. Henderson, L., A. Wolfreys, J. Fedyk, C. Bourner, S. Windebank, 1998. The ability for the comet assay to discriminate between geno-toxins and cytotoxins. Mutagenesis 13:89-94.
6. Visvardis, E.E., A.M. Tassiou, and S.M. Piperakis, 1997. Study of DNA damage induction and repair capacity of fresh cryopreserved lymphocytes exposed to H2O2 and γ- irradiation with the alkaline comet assay. Mutation Res 383:71-80
7. Fairbairn, D.W., P.L. Olive, K.L. O’Neill. 1995. The comet assay: a comprehensive review. Mutation Res. 339:37-59.
8. Collins, A.R., A.G. Ma, and S.J. Duthie, 1995. The kinetics of repair of oxidative DNA damage (strand breaks and oxidized pyrimidine di-mers) in human cells. Mutation Res. 336:69-77.
9. Singh, N.P., M.T. McCoy, R.R. Tice, and E.L. Schneider, 1988. A simple technique for quantitation of low levels of DNA damage in in-dividual cells. Exp Cell Res 175:184 -191.
10. Östling, O. and K. J. Johanson, 1984. Microelectrophoretic study of radiation-induced DNA damage in individual cells. Biochem Biophys Res Commun 123:291-298.
Acknowledgements
Roger Marine Biochemistry | ASU SMS
Contributors: Avery Kruger, Liza Roger, Enzo Life Sciences
Last modified: 06.08.2026