Recently we’ve started to set up comet assay in our lab. I've attached our microscopic captures from the slides we prepared as negative control (whole blood was used).
We would be very grateful for any interpretations/comments you can give us on these photos.
I dont think this picture is enough to comment on comet forming. I dont see any distinct nucleus with comment. You need to take picture at 1000 X. Please make sure that you take good care during staining and pos-staining washing. Because cellular material often lost at this point.
In your photographs it is unclear observing comets, it seems that is not working lysis buffer as it is not moving. Additionally seen very dirty, personally would be more careful in the preparation of gels and washing them.
Cheers
Dr. Carlos Alvarez Moya
Dear Ahmadi,
As already mentioned, there is no comet in the pictures and looks like dirt. In fact, looks like you focused other things, above the cover slip. It is hard to focus the material in the first time of analysis, but I think that you'll know if you see a comet, independently from the coloration.
If you have used fluorescent dye, you should see bright nucleoids or comets. I also agree with Kühnel that the background should be black in fluorescent analysis. I only know silver staining for bright field, and the photos suppose to have a bright background with dark comets.
Kind regards.
This image seems did not stain with any DNA dye, it is difficult to tell wether it is dirt or cells (although it looks like dirt). Try to use fluorescence dye such as PI or DAPI, it will help you to identify the layer which contains your cells. It is easily out-of-focus when you took pictures under bright field.
Another thing you should be careful is to seed your cells on a flat surface which will help all the cells stay on the same focal plane. A warm glass plate will help a lot.
Good Luck : )
Dear Nahid,
No comet seen in the photo. It has problems in preparing gels. Best regards
Thank you so much for your answers.
For visualization of DNA damage, observations are made of EtBr-stained DNA using a 40x objective on a fluorescent microscope. We thought the bright circles were comets and had no idea that we could have not black background.
Dear Nahid,
The absence of comet in the photos may be due to the preparation of lysis buffer, gels and washing. Moreover viewing them try using fluorescent dye DAPI or PI, which will help you identify the layer containing the cells. I suggest reading: Tice, R. (1995) “The single cell gel/comet assay: A microgel electrophoretic technique for the detection of DNA damage and repair in individual cells”, en “Environmental Mutagenesis”
(D.H. Phillips, S. Venitt, Eds.), Oxford, UK, págs. 315-39 Environmental Mutagenesis. pp. 315-39.
Best regards
Dear nahid, I've done comet assay for various type of tissue...luckily, based on method from journal we have successfully viewed the dna damage. So, in your picture, seems like the light is still on..
1.make sure the light source are switch off only UV light is on, choose green filter
2. try to view/focus at the edge of the gel first..by using fine knob..sometimes most of the cell accumulate at this area
3. keep calm and continue viewing..all the best..
fyi...we will organize a workshop on comet assay.. u r also invited
Dear Colleagues
Thank you so much for your help and time.
A few days ago I tried with another slide. Its photo was attached. Would you please help me if I had comets in this photo?(Sorry about its quality.)
Yours faithfully
If it is silver staining photo should be in black and white. It seems to be some what out of focus. But basically it is good work
Dear Nahid,
According to the photo be wanting a little tail, it is not displayed well. I think you need to improve electrophoresis. Best regards.
In order to obtain appropriate comets one has to standardize the methods in such a way that the sharp picture of comets are visible. For the purpose of obtaining the comets appropriately they have to standardize the slides with respect to human blood. as the control. The most important step is to maintain the electrophoresis voltage to 20 V (cc. 1V/cm) and current 300 mA. This will help develop optimum condition for obtaining appropriate comets.
Dear all,
Recently comet assay has been set up in our labs.
Thank you so much for all help and advice.
Sincerely,
Nahid Ahmadi
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