I'm planning to do comet assay for DNA damage using lymphocytes from rat. Does 1 ml blood sample enough if I plan to isolate the lymphocytes using ficoll-paque? If it is possible, do I need to adjust the ficoll volume (the literature often use 3 ml ficoll for 2 ml or more blood sample)?
1 ml blood is more than sufficient but you need to mix thoroughly with equal volume of warmed PBS and carefully load as a layer on warmed Ficoll. You should take care that Ficoll will not on the wall of the tube. After centrifugation you should wash the lymphocytes with PBS. Good luck
Dear Dr. Ian Teo and Prof. Ali Aldallal thank you very much for your help, Please don't mind if I ask further later. This is my first time using this method, and actually comet assay is uncommon procedure in my institution. Have a good day.
Best Regards,
Raesa Yolanda
1ml blood sufficient for lymphocyte isolation.... try to mix the blood with 1ml PBS and layer on Ficoll (1:1).. it will give good separation
1 ml blood is more than sufficient but you need to mix thoroughly with equal volume of warmed PBS and carefully load as a layer on warmed Ficoll. You should take care that Ficoll will not on the wall of the tube. After centrifugation you should wash the lymphocytes with PBS. Good luck
If you are planning to perform the comet assay immediately after you obtain the blood, you can directly perform the comet assay with whole blood
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