Hello! I am growing transfected cells in 24 well plate. on the bottom of each well i have a small glass cover-slip. so the cells adhere to that cover slip. I am using this method because its very easy to transfer that glass to a slide and then analyse for fluorescence. the only problem is that DAPI staining efficiency is super low. I am simply covering the glass on which the cells are growing with DAPI for 5 minutes and then analyzing. Is there another protocol that I should use in this case?
Thank you!
One of the reasons may be that you are using too much DAPI generally 0.5-1 ug/ml is more than enough. In high concentrations, DAPI quenches itself and counterintuitively you lose the signal. However DAPI doesn't penetrate into live cells very well, it is also harmful to the cells so I can't recommend it as nuclear counterstaining for live cultures. For the live cells, you can use Hoechst stain it is cell-permeable and much easier to work with. Just curious why you are not using fixation of your specimens? Mild fixation of PFA 0.5-1% for 3-5 min usually doesn't affect the GFP, RFP fluorescence much, you will have way more time to observe your cells, combine it with antibody satining and DAPI will work on fixed cells fairly well.
Hello Dr. Lennikov. Thank you very much for the answer. I was unaware that large amounts of DAPI is worse. I will try a lesser amount, Thank you!
DAPI staining is the final step in my experiment and after that I do not need live cells anymore. For that reason I am not concerned that it is damaging to the cells as I only need to take few good pictures. We do not have Hoechst in the lab right now so I thought I could get away with DAPI.
The reason that I am not fixing is because someone in my lab successfully stained the cells this way with DAPI and I am tying to replicate their experiment. I think the problem is the amount, as I am putting quite a lot of DAPI on the cells. However, I will try fixing as well.
Thank you very much!!
Dear James!
Please You look in following protocol:
https://biotium.com/tech-tips/protocol-staining-cells-with-hoechst-or-dapi-nuclear-stains/
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