Hi all, has anyone experienced their lymphocytes to disappear during the course of comet assay? We always count the lymphocyte concentration before embedding cells in agarose, but sometimes it happens that once the comet asay is performed and we put our stained slides under microscope we do not see any cells. Could this happen due to high agarose temperature or incorrect electrophoresis conditions? We always use 25V for electrophoresis. Thank you for any suggestions!
Dear Anna,
Greetings!!
By description of your problem, I think there is some issues with putting coverslips for uniform spreading of your agarose containing cells (here I am referring to the "second layer of agarose containing cells" which is between the first base layer and the final third agarose layer). Many times it happens that if you do not immediately put the coverslips then there is a tendency that agarose containing cells may get hardened and get stuck at some areas only, and are not uniformly spread all over the slide.
Another reason may be due to improper lysis of the cells, which might result in inappropriate staining later on. Make sure you are keeping your slides for a minimum of 2 Hours at 4°C, before proceeding for electrophoresis.
Hope it Helps!! All the Very Best!!
With Regards,
Samrat
I think that you should put another layer of agarose upper de gel with the cells
Hey Anna,
I had a similar experience during comet assay of lymphocytes. I think it was due to the absence of a 3rd protective layer of LMPA above the second layer containing cells and LMPA. Since then I always add a 3rd layer of 0.5% LMPA.
The following points can be taken care of to avoid disappearance of cells:
1. Even layering on grease free slides- the 1st later of 1% NMPA should evenly spread and dry completely over the slides.
2. Even mixing of LMPA with cell suspension (by pieptting) prior to layering over the base coated slides.
3. Gentle removal of cover slips after complete solidification of the 2nd layer containing the cells. The cover slips should be dry prior to mounting and immediately placed over the 2nd layer. They entire layer may come off while removing the cover slips if they are wet or if the layer does not solidify completely.
4. Use of a 3rd layer of LMPA. I generally use 0.5%. The same routine of coverslip mounting and removal is followed.
5. The pH of lysing solution should be maintained. I had observed that prolonged lysing beyond 24 h lead to loss of cells from the slides inspite of the addition of a 3rd layer. I usually lyse lymphocytes for 3 to 4 h.
6. Unwinding and electrophoresis- the pH of the buffer is crucial. Voltage fluctuation may lead to improper run.
I hope this helps!
Ilika
- Badges
- Science topic
- Similar topics
- Philosophy
- Philosophy Of Science
- Reasoning