Comet Assay - Science method

Good evening,

This is my first time experimenting with the comet assay. Specifically, we are running PBMC samples and using silver stain for visualization. Could this image observed under the light microscope (40x objective) be a comet that can be evaluated?

Thank you very much in advance!

I’m planning to run the Comet Assay to assess DNA damage, but I’m debating whether it’s worth investing in a specialized electrophoresis tank or if a standard tank can handle it well enough. Has anyone tried both? I’d love to hear any tips or experiences on whether the specialized tank really makes a difference in terms of accuracy or reproducibility.

good morning all

i have two groups to compare between them : control and persistence bacteria treated with antibiotic (X)... the DNA damage was estimated using comet assay ( Neutral )

what is the estimated from the parameters : olive tail moment , tail DNA % , tail length ??

i supposed that the control because untreated with antibiotic X should contain higher damage than persistence cells !!!!

any ideas

warm regards

I did comet assay with goldfish in my master's degree but now I am experiencing such a problem. has anyone encountered such a problem before? the only difference from my other experiments is that the LMAs have been in the ependorf in the refrigerator for a while and while taking blood, I cut the anal fin and put it on the pbs from the cut point and let the blood flow into the pbs. there was no difference other than these two.

Thank you in advance for your assistance.

My negative control cells had some damage, they are neoplastic cells and my anaplastic cell had even more damage in the negative control.

Ok, but my question is, is there much difference between the controls? For example, there was one of mine that had 6% damage in the negative control, but 17% damage in the positive control. This is weird? Wasn't the positive control supposed to have a lot of damage? Or does this not apply?

Another question, is the positive control just for us, to see if the experiment worked, or does it need to be included on the graph?

Thanks!

Hi everyone

it is possible to freeze bacteria cell with saline under -80C for next day ( for e.g) to start comet assay ( determine DNA damge ) !!

Or

I should use fresh prepared suspension bacterial cell ?

thank you

Iam trying to optimize the Comet assay protocol for Yeast cells in my lab but iam unable to lyse the yeast cells with the regular protocol and hence those cells are seen intact even after the treatment. I looked upon some protocols which suggested the use of Zymolyase/ Lyticase for the Yeast cell wall lysis, but my lab doesnt have either of these enzymes and hence can someone suggest any other enzymes apart frombthe above mentioned that I can use for the same? Also can I use Lysozyme for digesting the Yeast cell wall?

Is Low melting point agarose and agarose having low EEO the same? Is it possible to utilize agarose low EEO instead of low melting point agarose for COMET assay?

The currently known methods for detecting DNA damage, such as the comet assay, are generally applicable to all DNA. I would like to inquire whether there is a specific DNA detection method for a particular gene? Thank you very much for answering my question！！！

I am doing comet assay for gills of fish, the fish were exposed to pesticides primarily.The problem i am facing that the cells are not picking stain.Can anyone tell that while using acrydine orange in order to stain DNA of gills.which concentration we should use?

comet assay

comet assay

Hi, in a few months I will have to do a comet assay by taking blood sample from people as a sample. The problem is that I don't know anything about it, so I wonder if there is someone who can help me and tell in which book I can find information and protocols about this.

I am interested in doing comet assy on arachnid spermatozoa, and I would like to know the best protocol to carry it out homemade. For example, which slide is suitable, how to adhere the low melting agarose, which buffers to use to make the cell suspension, etc.

the mA and vol in the paper did not get a good running

I did prepare 1litre of comet assay lysis solution by adding sodium lauryl sarcosinate. For the assay used only 100 or 150ml and kept the remaining at 4 degree celsius for future use. After 2 days, white crystal precipitate has started to appear.

We did comet assay based on joves protocol. Under fluorescence microscope, cells were visible but without tail. The drugs showed toxicity when grown in 6-well plate. Lysis was done for overnight at 4 degree celsius. Electrophoresis was run at 25V, 300mA for 30mins.

I'm currently searching for comet assay kit, which can give almost accurate results and should be cheap of cost. Can anyone recommend any kits regarding this that you have worked with?

Many articles are suggesting CASP software to analyze the comets. Please share the details if there is any free access to this software or any other.

Thank you for coming by.

For preparation of lysis solution for comet assay we needed tris base. But we found tris buffer on our stocked. When searched on internet both were having same formula.

Please anyone send me the link for software of comet assay gel electrophoresis analysis.

I performed several Neutral comet assays using Trevigen protocol . What I have noticed is that whenever i used HU( hydroxyurea) positive control and for my transfected cells(1mM, 2mM and 5mM) , there has been a huge halo formation around the head of the comets which makes it very difficult to analyse using Opencomet software. I think it might be an effect of HU because, I don't see it in untreated and neither do i see it when i do neutral comet assay with Camptothecin. Has anyone experienced this? If yes, what could be the reason? Any suggestions?

As per the standard protocol set of comet assay, we had prepared the slides and done the Lysis procedure for 1hr.. later horizontal electrophoresis for 30 min in stationary phase and 30 min run..for 300mA and 25V.

But still all the DNA comets are not moving in one direction, their direction is not fixed , what may be the cause or is it ok.. to analysis the same..

Blood sample: Chronic kidney disease (CKD) patients

Dr.Kiran Nikam

Asst,Prof,

BIMS, Belagavi

I am working with U87mg cell line. After exposing the cells with the dose for a time period. I have to harvest the cells for comet assay. How can I store these cells so that I can perform comet assay later. Can I store it in PBS and at what temperature ?

I am treating my cells with a drug that generates about 20-40 uM H2O2. I see a profound activation of gamma-H2AX but no/very low levels of corresponding DNA damage in comet assay.

I am trying to find out if the induction of gamma-H2AX is somehow related to the generation of H2O2 in my drug treated cells.

I've performed the alkaline comet assay on isolated zebrafish cells, and sometimes I find some nucleoids with this "granular" appearance, without forming a tail, both in negative control and treated cells, at the same time that I find " normal " nucleoids (red box of the image). My positive control is working well, using the same cells, so I excluded that it is a problem with electrophoresis. Before starting electrophoresis, I leave it in fresh lysis solution (2.5M NaCl, 10 mM Tris, 100 mM EDTA, 1% Triton X-100, 10% DMSO) for 1h30, and in unwinding (300 mM NaOH, 1 mM EDTA ) for 20 min. Has anyone seen nucleoids with this appearance? Any tips?

*attached are some images of these "granular" nucleoids, in red box there is a "normal" nucleoid from the same treatment.

I started using Abcam comet assay kit and I have several problems regarding agarose application. First, agarose doesn't spread well and doesn't cover the well during the initial base level creation and then during agarose+cells mixture application. It actually forms a drop of a sort, thicker in the center of a well and thinner on the edges. Should I use a coverslip to make the layers even?

The other problem is the gel falling of the slide during the first wash after the EF, even though I apply the 1V/cm voltage as mentioned in protocol. The gel stays in place before the EF. What can be the possible reason for that? May be I should use cold buffer and perform the electrophoresis at 4 C.

Thank you!

Hi!

I am currently optimizing a protocol for the comet assay in my lab and I was wondering if I could use a regular microscope slide for the assay? I tried coating the slide with one layer of 1% NMP agarose but I do not see the layer. Should I double coat?

Also, since they are regular slides, I do not have the circle shaped wells on them to drop my cells. Once I add approximately 75 ul of sample mixed with LMP, should I spread the drop or leave it as it is?

Any comments and suggestions would be appreciated!

Thank you !

I am working on doing a comet assay and I am using the Trevigen kit. I was wondering after you add your SYBR gold and do the quick rinse in water. Can you just leave the slide to dry in the 37 C incubator overnight? I've been having trouble with blurry slides and wondering if it's left over agarose I'm not seeing.

Thank you

Comet assay for plant cells

We are working on comet assay experiment in single cell electrophoresis using bromide ethidium. We have ruled out cell absence in the samples and there does not seem to be any problem concerning the microscope, so after many efforts we are wondering what might be wrong. Do you have any suggestions for agarose techniques or special indications on the experiment?

Thank you in advance!

I am new to comet assay and I seem to have trouble with the slide. When I go to image it I can't focus it and is seems blurry. I have checked to make sure that it was not the microscope. Anyone else have this problem?

Hello.

I have conducted the comet assay for the genotoxicity test.

Last week, I did an experiment comet assay and found something strange about the comet image.

Not all slides are like that, some of the other slides tested at the same time have a normal shape.

What is the reason?

Attach both images.

Please accept my thanks in advance for the favor of a reply.

I am using Comet Analysis as part of my thesis and after having used Cell Profiler (can't find the head), OpenComet (misidentifies the head and created outliers) and CASP (does not work with windows 10) they have all failed to pick up even the cleanest data that I generate.

From a publication perspective, is it acceptable to still use manual classifications for comets in this day and age of technology? I find this method very labour-intensive, but it is very reassuring to know that all my comets are classified correctly and not turning into outliers because of unreliable tech.

I plan to have 3 to 5 biological replicates and use confidence intervals instead of p-values to show consistency.

Hi, everyone. I have been conducting comet assay for analyzing the genotoxic effects of materials.

My problem is that the background of the microscope image is all green. So that I can`t observe my cells. Please tell me what should I do.

I have many hands-on experiences in carrying out Comet Assay method on various animal cells, but I haven't had any background to investigate DNA damage in aquatic plants so far. As they are single cells in their populations, it seems to me such cells possess a merit to conduct DNA damage studies (single cell gel electrophoresis). However, I don't know how I can conduct an Alkaline Comet Assay procedure on either marine or freshwater microalgae? especially when I need to remove a plant cell wall which is more harder than the animal one (lysis step) .

I really appreciate it if you attach instructions and related papers.

Many thanks

Milad

How do you adjust the base layer thickness of agarose gel when electrophoresis in a comet assay?

When I was doing peparation of Slides, dip conventional slides and wipe underside of slide and just lay the slide on a flat surface to dry.

I think it will be a little bumpy even if it looks flat and thin to the eyes.

In this situation, does the thickness of base layer affect electric motion?

have a safe and healthy days

i want to know what is the best solution to be used instead of HBSS solution in comet assay while using g bone marrow samples

thanks

In several repetitions that I performed, there is no tail formation on the nucleoids, even using different mutagens / damage inducers (MMS - 200 uM; H2O2 - 400 uM and Cisplatin - IC50 value). I have already performed the comet assay with hepatocytes from C57BL6 mice and with A-375 and B16-F10 cell lines and this problem still persists.

The running buffer used is: -H2O (Deionized water) -NaOH 10N -EDTA 200mM pH> 13

25V / 300mA - 20 minutes

**During electrophoresis, there is the formation of bubbles in the electrophoresis buffer (I believe the electrophoresis chamber is working).

The concentration of agarose used was:

Normal agarose - 1% or 1,5% (v/v)

Low Melting Point Agarose - 0,5 % or 1% (v/v)

I appreciate all the help available

*Attached are some images of the nucleoids that I obtained in my last experiment (A-375 Cell line).

Hi everyone, ı have a study on Eisenia fetida. I determined the LC50 of a chemical on E. fetida. want to perform comet assay with the concentrations LC50 and 2xLC50 but all the e. fetida animals are dead in 2xLC50 concentrations. Can ı perform comet assay wtih these dead cells?

I'm working on MDA MB 231 cell line, which is adherent cell line, I have read a couple of articles that collect cells by trypsinization that some resuspend in complete media and others in PBS, which is best?

and another question,I'm trying to optimize the best dose and dose exposure, hence I'm doing several ( Time exposure) what if I freeze those pellets down until all of them are ready so I can do the essay at once.

want to know the preparation of the liver samples for comet assay

I am currently doing alkaline comet assays on a HaCaT cell line. I'm fairly new to comet assays. I would like to ask opinion about the result I obtained for my experiment. So I run untreated (picture 1 and 2) and also positive control with H2O2, 200 uM for 30 minutes (picture 3 and 4).

I expect that comet tail should appeared for positive control. But somehow, from the picture, I cannot identify any comet tail.

I would be very thankful if someone can clarify the problem, and whether I’ve done it correctly or vice versa. Thanks!

Slides for the use of Comet assay?

Hi everybody,

could someone suggest me how long i should incubate cells with h202 and at which concentration to obtain positive controls? Moreover i don't understand how should i perform statystical analysis of resuts obtained (tail lenght , tail moment , percentage of dna after treatment with increasing concentrations of a drug) in view of the fact that the means of each conditions are not omogeneous. Is it a good strategy to transform my data in log scale? what is the right post hoc analysis and graphical representation?

many thanks

Francesca

Is the cost-benefit positive enough to acquiring kits even though they seem to be more expensive than buying the reagents separetely? We will perform a validation of a product using this method, and I'm concerned that the investment at this point might not be ideal since we are still otimizing the protocol to a specific laboratory setting.

If you do find kits a more suitable choice, could anyone recommend me a brand?

Thank you in advance.

I'm treating my cells with a drug inducing SSB rapidly converting into DSB. While I'm able to measure total level of DNA damage via Comet assay and all kinds of yH2AX, is there a specific DSB DNA repair assay? I know there are kits for BER and NER, but not aware of other types. Thanks

Can I substitute low EEO for low melting point agarose when running a comet assay?

Dear senior's i'm working on comet assay but unfortunately i didn't find any free software for comet analysis.if someone have software like CASPlab, open comet, comet !v etc then kindly send me on

abdullahbiotech2@gmail.com .

thanks for anticipation

Dear senior's i'm working on comet assay but unfortunately i didn't find any free software for comet analysis.if someone have software like CASPlab, open comet, comet !v etc then kindly send me on

abdullahbiotech2@gmail.com .

thanks for anticipation

I have understood that analyzing genotoxicity with the comet assay depends on which parameter I decide to use, and according to some other publications & scientists, some of them prefer one over another. But, I would really like to know what are those preferences based on.

I'm currently trying to determine the genotoxic effect from some synthetized molecules but I have differences in the results between these three parameters. I’m having trouble on understanding which one would provide me more accurate results or if it’s ok if they all are not related in those three parameters. I would really appreciate some guidance and responses.

how can DNA damage be assessed in fish exposed to toxicants order than comet assay?

hi, does anyone have idea upon measurement of results of comet assay without using a specific software? alternatively could anyone suggest me a free software for quantification of my results?

Same as the title.

With alkaline electrophoresis buffer, power supply do not run.

(no voltage on screen, no air bubbles)

But, it works well with TBE buffer.

I want to use alkaline buffer with my comet assay, but I cannot find relevant trouble shooting method despite of rigorous searching.

Could anyone help?

Hi.

I am working on a research on buccal cells using comet assay.

I tried open comet and Casp software but there is no match between results on the same comet . Moreover, these soft ware consider artifacts as a comet .!!

I need your help to find software that I can trust in. or if you know a company that analyse a set of pictures of comets.

Best Regards.

I've been trying to optimize COMET assay using the standard protocol. I tried with EtBr and SyBR gold stain but couldn't get any fluorescence.?

Dear comet assay community,

Does anyone ever see dehydrated or crystalized gel after slides drying?

On the picture, the left slide is what our regular comet slides look like and the right slide is the problem we encounter for the first time. Some slides look dehydrated or crystalized after 3 days of storage. The same assay solutions were used for both slide batches. And as usual, we end up the assay with an ethanol bath.

Any explanation for that?

I used Oxiselect comet assay slides but followed the Trevigen protocol. Is there a way to preserve the stained slides for few months or even weeks? Will it be enough to store them in the dark at room temperature?

Thanks

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:

Day 2:

Day 3:

Day 4:

I would like to do comet assay in heavy metal treated fish. Can anyone help me to find reference materials for it?

I am struggling with getting quality results from my COMET assays, and have been using DAPI as a stain. Is there any reason DAPI can't be used? I'd rather not use EtBr but will if DAPI is the issue.

The attached pictures are all cells that have undergone the same treatment in the same agarose gel. Why is there so much variability?

My protocol is as follows:

-Resuspend pellet in PBS.

-60min in Lysis Buffer at 4 degrees Celsius in the dark

-30 min in Alkaline Electrophoresis Buffer at 4 degrees Celsius in the dark

-40 min electrophoresis at 1volt/cm, 300mA current

-Dehydrate with 70% ethanol for 5 minutes, airdry overnight

-Stain with DAPI

i want calculation damage index with five comet index includes:comet lenght, comet height, tail length, %DNA in tail and tail moment.

I ask about the names of stains used in comet assay ,can any one help me?

Comment on the validity of comet assay method for DNA damage in organs and tissues. How to reduce the error compared to blood cells?

In my attempt to do NEUTRAL Comet Assay using HCT116 cell lines, my control (untreated) cells always show significant comet tails compared to the positive control group. I used Doxorubicin and H2O2 treatment (separately) as the positive control group. Samples were harvested by trypsinization and collected in ice-cold PBS before mixing with 0.7% low melting point agarose. 1 hour lysis in 4 degree Celcius was done using the following lysis buffer ingredients: 2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, 1% sarcosine, 0.5% Triton X-100, 10% DMSO final, kept at 4 °C.

Electrophoresis was done in pre-cold TAE Buffer at 18 V (0.8 V/cm) during 30mins. Samples was treated in the dark throughout the lysis until visualization.

I have tried different voltages and even used TBE for electrophoresis yet my control samples still show the comet tails. Can anyone give suggestions? Thanks!

Have anyone used the WST-1 assay for ID8 cells?

Is 1000 cells a good starting amount to seed them? 

Can I induce cell death to my cells using Triton X and then use the WST-1 assay after to measure viability? Because I read that Triton X is used as a stopping solution, so I don't know if it will interfere with the reaction. 

Dear Investigator,

The comet assay has been in existence for many years, but the general protocol largely remains unchanged, despite it requiring numerous, time-consuming steps. We in the Oxidative Stress Group, at Florida International University, are very interested in learning about comet assay users’ experiences, and their thoughts on the protocol. Below is a link to a short questionnaire that we would be grateful if you would complete. All replies are entirely anonymous, but you will have the opportunity be entered into a prize draw for an Amazon voucher.

https://fiu.qualtrics.com/jfe/form/SV_dh9MYqcgHp42LoV

(you might need to cut and paste into browser.)

Feel free to distribute to other colleagues who may be interested.

Please feel free to contact any of us if you have any questions, or want to confirm the veracity of this email and questionnaire.

Thank you very much in advance,

Marcus

Professor and Head of Department.

Oxidative Stress Group, Dept. Environmental & Occupational Health,

Florida International University, Miami, FL 33199.

I would like to compare the DNA damage at basal level (without any treatment) between a cell line that lacks the expression of one protein vs the parental cell line. I have read that the comet assay is a simple and rapid technique that allow to see if one cell have some defect in DNA repair (single or double breaks) but I am not sure that is the right technique for my experiment or I should search for a different approach.

Any suggestion would be very appreciated.

Thanks!!!!

I have tested radiosensitivity of drug-treated cells and radioenhancing effect of heavy-metallic nanoparticles for a while. It seems that clonogenic assay, gamma-H2AX assay, comet assay (or DNA ladder) and micronuclei formation assay can be used for the evaluation. Does anyone know which one is more validate or any notes of each assay for different applications? Thanks.

Is it possible to reuse/remelt the agarose (low gelling temperature) after it has solidified ?

In comet assay, Could you suggest to me, the cells can be used until which passage?

1) I have problem with electrophoresis power supply, when I set voltage to 25 V, mA only reached to 120 mA. To make 300 mA I have to increase voltage up to 45. (I also run electrophoresis at 4°C) Does it effects the results negatively? (in general protocols I read recommend 25V, 300mA)

2) Also I attached my positive control (H2O2) images. I consider that my gel is damaged what do you think? I precoated my slides with 1.5 % Normal Melting Point agarose after mixing the cells with 0.5 % Low melting point agarose (20ul cell/ 120ul LMA). What should I do to improve my results?

I'm waiting for your recommendations

Thank you

I have performed multiple comet assays and have been using the imageJ plugin OpenComet (version 1.3.1) to analyse the results. After running through the software and viewing the images with the software overlay, I have encountered a number of instances where the software has not picked up the presence of a comet. I know that the software flags any events that it cannot analyse due to irregular shape or overlapping of more than one cell etc. and outlines them in grey. However this is not what I am referring to as the software has appeared to ignore the presence of a number of comets with very normal shapes, characteristic of a comet. I have included several cropped versions of images I have taken, focusing on the undetected comets as examples.

In image 1 there is a very nice comet in the top left corner. In image 2 there are several comets on the left side that went undetected. In image 3 there are a number of comets that the software did not pick up, particularly in the right half of the image.

Can anyone provide me with some clarification about why this might be happening? Or any potential solutions?

Many thanks in advance.