COMET assay gone wrong, how can I interpret these results?

02 February 2019 4 5K Report

I've performed a COMET assay multiple times, improving on the procedure each time and re-making my solutions, but continue to get the same inconclusive results. My supervisor and I both tried refocusing the microscope but it does not appear to be able to focus on anything beyond what is imaged. I am trying to interpret what I am seeing, and where I went wrong in my COMET assay. It does look like a few COMET-like forms are in some of the photos, but it seems wrong because they are in all different directions.

Any thoughts, suggestions, or advice would be greatly appreciated!

Thank you!

I've added a simplified version of my procedure below:

Day 1:

Prepare cells to be used. Cultivate cells for 24 hours in 6-well plates.

Pre-coat microscope slides with 1% agarose (improves adherence).

Day 2:

Add the treatments to the wells and allow 24 hours to take effect.

Day 3:

Add 80 uL of the cells and 240 uL LMP agarose to an eppendorf tube.

From this mixture use a micropipette to add 2 80 uL drops onto the pre-coated slide. Immediately add cover slips. Place in fridge for 5 minutes at 4°C to solidify, then remove the cover slips.

Prepare lysis stock solution with NaCl 2.5 M, EDTA 100 mM, and Tris 10 mM - pH 10

From the lysis stock solution, add Triton X-100 and DMSO to make the lysis solution.

Place slides inside a copplin jar with the lysis solution and keep overnight at 4°C.

Day 4:

Prepare electrophoresis solution (EDTA 0.25 M and NaOH 300 mM with cold distilled water - pH=13)

Pour into electrophoresis tank.

Place slides inside the tank with the solution for 20 minutes.

Run the electrophoresis for 25 minutes at 25 V.

Inside a copplin jar, place slides with neutralization solution (Tris 0.4 M with distilled water - pH = 7.5) for 30 minutes at 4°C.

Fix the slides with absolute ethanol for 5 minutes.

Stain slides with a few drops of DAPI.

Count the cells with a fluorescent microscope

Prachee Gokhale

I had this once - comets in different directions. When you place slides in the electrophoresis chamber, ensure all slides are orientated in the same direction. For example the frosted end of each slide facing right. My chamber can hold a total of 12 slides in two rows of 6 each. In both rows, I place the slides such that the frosted end of all slides is to the right.

Once I corrected for this, I have had not had this issue again. Hope this helps.

Maksym Kitsera

Dear Brenna Hay,

We perform Comet assay differently (if you want zou can check here - Article Klebsiella oxytoca enterotoxins tilimycin and tilivalline ha...

But I immediately can give some suggestions :

1)It is no need to leave slides in lysis solution overnight just 1h should be okay.

2)Ratio between cells and LMP should be from 1 to 6 till 1 to 8.

3) It is also no need to use cover slips, with this one you still can damage cells.

4)Instead of leaving slides for 20 min. you can use Alkaline solution (just 3 g of NaOH in 100 ml of water)

5) again it is no need to neutralize electrophoresis solution, just wash slide with water 3 times for 2-5 min.

6) All, Slides should be fixed during electrophoresis.

Brenna Hay

Thank you so much, I'm giving it another try in the next week or so and hopefully get better results!

Blessings Hendrina Likoswe

Hello Brenna,

Where you able to get past the hiccup? We are running into similar problems where our slides are just as unclear as yours (some with comet going in all sort of directions). The protocol we use is similar to yours so maybe you can provide some pointers? Our images also look quite similar to yours.

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