I am working with NIH 3T3 cells at the moment and I want to do live/dead assay. I use Calcein-AM and Propidium Iodide. I have done several trials so far. However, no matter what I do I keep observing that my Calcein + cells are also PI +. IN addition, the staining looks very nice, Calcein is cytoplasmic while PI is nuclear. I have done titration of the concentrations for both Calcein and PI. Tried staining them simultaneously or separate from each other. Did anybody else experienced such a thing? I just can not explain what is going on?! Thanks very much.
Hi Gozde - it may be that what you are observing is "bleed-through".
i.e. you Calcein staining is so strong that you are picking it up in you "red" detector.
(calecin labelling can be very,very bright is some cell types).
Try reducing you labelling time by 50-75 % and see if you get the same result.
Also, if your microscope is capable you could check this by exciting at 488 nm and look at the channel for PI emission - if the cell appear to be "red" after 488 nm excitation this is almost definitely the "green" bleeding through to the red channel.
PI can detect RNA as well as DNA so it gives a general "cytoplasmic" as well as "nuclear" signal which is similar to Calcein (so bleed-through is indistinguishable).
You could also swap to Ethidium Bromide as this gives a specific, nuclear signal.
Hope this helps,
Gary
Hi Gary, thanks very much for your answer. After I have posted my question here, I also realised it was the Calcein bleeding into PI channel. I have tried several things but the ultimate solution was doing imaging in multiple track. Thanks again.
Best,
Gözde
Hi Gozde,
I am having similar issue. Would you please elaborate a little bit how you sorted it out? Thanks in advance.
Hi Gozde,
Could you share your experience about solving the bleeding issue. As I still see a little bit overlapping in Red channels with green one, after trying different things like decreasing staining time, different excitation wavelength, reducing the laser gain,..
Thanks
Is this problem because of the high Calcein AM concentration used? Gozde Akdeniz Skvortsov how were you able to sort the issue, could you elaborate?
I was able to sort out this issue by reducing Calcein AM concentration to a nanomolar range which reduced bleeding. You have to play with the laser intensity to get a clear idea about the dead and live cells.
Thank you for all your comments, they are very useful for me.
I have the same problems when I encapsulate cell into an hydrogel. But, what I do not understand is why the area of staining is different in green and in red. What I mean is that the red staining seems to concentrate in a small area that could correspond to the nucleus, because it is like a spot. Meanwhile, the green staining is a broader area.
Thanks your in advance.
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