Have some basic queries regarding initial setup:
1) Do I need to use the microscope in fluorescence mode?
2) If I put my stained slide on the stage, will the image on the screen remain black, or show me fluorescent cell nucleus?
Any tips will be greatly appreciated!
Dear Jhoti
There are many protocols for performing Comet assay.
You need to answer some questions for choosing most appropriate one.
1- What kind of cells you want to test DNA damage?
2- What is your specific or potential stress( or pollutant)?
3- What is your scoring criteria?
4- What is your staining method?
If you use ethidium bromide for DNA staining, you need fluorescent microscope. If you use silver nitrate for DNA staining, you need light microscope.
In ethidium bromide staining, the comet image has dark background and you see the comets in the shining form.
I think the attached paper is very helpful. I have performed comet assay on brown trout with silver staining method.
Best wishes
Mehran
Thanks for replying!
I'm using SW872 and 3T3-L1 adipocyte cells. I'll be inducing oxd stress with H2O2 and i'll be using either EtBr or acridine orange.
From your answer, it seems that my microscope should be set to fluorescent mode. the cells will appear like white dots?
I think you will see the comets as relatively shining dots and strings
Dear Jhoti,
If you'll use EtBr, the comets will appear colored red/orange direct on the objective lens. If the image is captured, the colour will depend on the camera used (coloured or monochromatic - the last one will present bright white comets). See the figures attached to compare. Furthermore, you'll need to use the proper microscope's filter. If I'm not wrong, the filter for EtBr is based on the wavelengths around 510 nm (excitation) and 590 (emission).
There are protocols that use silver staining, but it leaves a dirty background in the slides which can disturb the measurement of the tail. In the other hand this technique allow you to analyse in a normal microscope.
Hope this help you!
Good luck!
Dear Jhoti,
I forgot to mention that if you intend use EtBr, be careful, because it is very toxic. In our lab we've changed it for GelRed because it uses the same microscope specification and it's more safe.
Best regards!
Matheus and Tanya, your advice is highly appreciated. Thanks very much.
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