We suspect mycoplasma contamination in one of our cultures since DAPI staining reveals cytoplasmic staining. But one person suggested it was simply mitochondrial. This got me thinking: in all stainings we have done and in all staining I have seen published where the investigators performed DAPI staining to reveal the nucleus, why do we never see mitochondrial staining? It should reveal many cytoplasmic structures yet we don't see any.
Agree with most of the comments above. It is like very difficult to see stars during the day time since the son is too bright.
I think you usually do not detect it because it is not enough to give sufficient signal. Most cell lines do not have enough mitochondria or mitochondrial DNA to be able to detect it with DAPI. I have only seen it in one cell line, HK2 renal tubule cells, which are loaded with mitochondria, while they were found negative for mycoplasma, by three different methods (PCR, ELISA and a TLR2 based system). My guess is that you could also detect MT DNA with DAPI in other polarizable epithelial cells like MDCK.
On the other hand, I also hapened to detect this in beating rat neonatal cardiomyocytes, before they could get infected by mycoplasma.
In any case, there could be other reasons, like MT membrane permeability.
Perhaps DAPI does bind to mitochondrial DNA. But mtDNA unlike human DNA is not tightly packed into compressed nucleosomal structures. So perhaps the little staining observed for the circular mtDNA contributes towards background.
You don't detect mitos with DAPI as actually the nucleus is far too bright in staining to see them. In addition, it could well be that DAPI is not easily penetrating the inner membrane of the mitochondria, but that is pure speculation. Binding itself to mtDNA shouldn't be a problem.
I don't see why DAPI should not bind mtDNA. However the signal intensity is uncomparably higher for nuclear DNA and that is my guess why you don't see the fluorescence of mtDNA in your cells.
No its not like that. You can use DAPI to stain mitochondria as used in these publications.
Article Detection of mitochondrial DNA in living animal cells with f...
Article Mmm1p, a Mitochondrial Outer Membrane Protein, Is Connected ...
Probably just your microscopy or camera configuration. DAPI signals in organelles are evident in cell staining, but in lower range of signal intensity. DAPI also stains RNA and prob RNA-rich organelles. Organelle band (plastid and mitochondria combined) show prominently in plant meiotic cytoplasm -see Fig. 2L-O in Yang et al., (plant cell) mmd1 Arabidopsis study for ex.
http://www.plantcell.org/content/15/6/1281.full
DAPI will stain mitochondria but the size of the DNA and organelle are so small compared to the nucleus that it is frequently not noted. An example of how small DNA structures can be detected by DAPI is in examining .22 um filter sea water - a pattern of a myriad blue dots of viruses are seen.
You always see mitochondrial DNA using DAPI or using any other interchelator.
Typically people remove most of the signal during 'background' subtraction, although it is always there.
In Trypanosomes (have only one mitochondria) mitochondrial DNA is packed in concatenated maxi and mini circules and DAPI stains mitochondrial DNA. I think is a matter of size.
Also, there ir a PCR method to detect mycoplasma.
Hope it helps.
Agree with most of the comments above. It is like very difficult to see stars during the day time since the son is too bright.
I agree with both of the above, and like the idea of counterstaining to see where the mitochondria are. The mitotrackers are very easy to use - I've had very good results using mitotrackerROS, for example.
Mitochondria are not laded with DNA, and the dye might not permeate into mitochondria either.
As most people say here already, the nuclear signal being brighter is probably the reason. Another possibility is the exposure time. You probably don't need too high an exposure time to visualise the nucleus, and may be a higher time is needed to see mitochondrial DNA.
DAPI will stain cellular mitochondria. It is possible to visualize the organelle if a long enough exposure time is used while fluorescence imaging. When DAPI used for nuclear imaging, usually the exposure time is too low, which is insufficient to pick up mtDNA staining. Moving to a field away from the nucleus will also help in imaging mitochondria without the field getting saturated with signal from nucleus.
I have seem mitochondria in DAPI tissues infected with Rhinosporidium seeberi. They appeared as very small dots.
I'm working with spermatozoa. Mitochondria, in these cells, are located exclusively in the midpiece of the tail. When I use DAPI, only the head of the sperm is stained.I have never seen the mitochondria of the midpiece stained with DAPI. Hope it helps
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