I am working on plant extraction and I want to check the DNA protecting activity of my extracts. How does the comet assay work? Is there any other alternative to comet assay? Can anyone provide or suggest a detailed protocol for the comet assay? Which cell line is required to perform comet assay? Can I use lymphocyte for comet assay?
How can I calculate the score and what is the interpretation of that?
Yes you can use lymphocyte for comet assay. Comet score software is available (free version) to score DNA Damage.
We have used the Comet for various compounds including some extracts. Isolated lymphocytes are usually employed as surrogate cells to test for DNA damage/integrity. I would recommend to get the Book "The Comet Assay in Toxicology" which will answer most of your questions regarding the Comet assay (http://www.rsc.org/shop/books/2009/9780854041992.asp).
Dear Adi,
Thanks for advise I will get the suggested book. Can you share the detailed procedure of lymphocyte isolation.
With regards
Dear Devendra,
You can use any lymphocyte separation procedure (e.g. from Sigma). We use Lymphoprep from (Axis-Shield, #1114547).
This is the protocol we use:
- mix 1 volume of blood with 1 volume of saline
- carefully overlay 6ml diluted blood on top of 3ml Lymphoprep. Do NOT mix the two layers!
- centrifuge at 800xg (e.g. for Mistral @1900rpm) for 20min at RT
- carefully remove the white lymphocyte layer on top of the Lymphoprep with a plastic Pasteur pipette. Do NOT disturb the Lymphoprep layer.
- transfer the lymphocyte layer(s) into a Universal tube, pre-filled with 10ml saline and invert tube.
- centrifuge at 500xg (e.g. for Mistral @1500rpm) for 15 min at RT
- discard supernatant and resuspend pellet in 900μl FBS
- transfer 450μl lymphocyte suspension into 2 cryo-vials each, pre-filled with 50μl DMSO and mix
- freeze all cryo-vials at -20C overnight then transfer them into a -80C freezer for storage
- defrost at 37C before using the cells for the Comet assay.
Kind regards.
Dear Adi,
Thank you for sharing your protocol, I ill follow your instruction for my future experimentation.
Sincerely yours.
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