Hello,
I am performing immunofluorescence of HepG2 cells. I believe that some of my stocks may be contaminated with Mycoplasma because when I stain them, their membranes and portions of their cytoplasm are blue as well as the nuclei. Is it possible that I have simply over-incubated with DAPI to produce this non-nuclear signal? Also, why is DAPI unable to stain mitochondrial DNA? Is it unable to permeate mitochondrial membranes? These membranes should also be opened by Triton X-100, correct? Also, I should add that in the case mentioned above of membrane staining with DAPI, I reduced my Triton-X-100 (0.5%) incubation from 10 minutes to 1 minute because I am attempting to visualize a membrane-bound protein.
Thank you!
Mitochondrial DNA by DAPI, it does but size is so small in comparison to nuclus: The question has been discussed here:
https://www.researchgate.net/post/Why_does_DAPI_staining_not_detect_mitochondria
Hey David,
I would recommend a mycoplasm PCR to test your hypothesis. Otherwise you would not know if your extra nuclear staining is from bacteria. The cellular staining can of course be the result of stained mito-DNA
David,
DAPI staining in the cytoplasm and associated with membranes can be a sign of mycoplasma contamination. There are other explanations, however. DAPI can stain DNA and mRNA, although its emission peaks are different, 460nm for DNA, closer to 500 for RNA, so if you have too much staining and non-specific imaging conditions, that could be a possibility.
DAPI does need permeabilization for the consistent crossing of membranes whereas something like Hoechst 33342 is completely permeable. BUT staining of mitochondrial DNA is quite challenging as there is so little of it compared to nuclear DNA, if you could see the mtDNA, the nucleus would be massively overexposed in the same image. Mitochondrial DNA is seen more easily with Pico green for example, or by staining for the TFAM protein as a marker.
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