I want to check c-value of dinoflagellate sp. using flow-cytometry.
Flow-cytometry machine in our laboratary can't detect blue fluorescence well, so I used PI which has red fluorescence.
Drosophila nuclei stained with PI was detected clearly, but dinoflagellate cells were not stained.
I had fixed with methanol:acetic acid(3:1 volume) and washed with 90%methanol.
And then, samples stained with PI were buffered by Galbraith buffer.
What is the problem? I guess that the thecal plate of dinoflagellate wasn't degraded. So, now I'm looking for method how to penetrate the cell wall. Is it the right guess?
PI only penetrates dead cells. You can first fix the dinoflagellate (with glutaraldehyde 0.1% for example).
If the dinoflagellates are photoautotrophic, chlorophyll fluorescence will interfere with PI fluorescence and cause an overestimation of genome size.
Regards,
Claude
You can try this method:
Fix cell with 70% ethanol, washed with PBS，then stained with PI .
I've never worked with dinoflagellate but I believe the ones which have theca might be resistant to detergent mediated permeabilization. I've found this article that uses an enzymatic cocktail to permeabilize. Maybe you should try it.
http://aem.asm.org/content/74/7/2244.long
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