I'm now needing to image cell cultures. I am thinking about transiently transfecting a fluorescent DNA marker, but I think it will be too sparsely labeled for my purposes. And throwing a dye on would be simpler and would let me know if more rigorous methods were called for.
Does anyone have a reccomendation for good live nuclear staining dyes?
Hi Philip,
I think ortho-Toluidine blue, my favorite dye, may be used for this. In the past I also used acridine orange to discriminate between living and dead cells as it has distinct colors depending on whether it is bound to double stranded DNA (green) or RNA (orange). Perhaps this article will be helpful:
http://cshprotocols.cshlp.org/content/2008/7/pdb.top50.full
Kind regards, Leo
I assume you mean a fluorescent dye. Others here suggest Hoechst 33342, which is excellent in blue. If you need green, I suggest SYTO 9. Red would be SYTO 59. Far red DRAQ5. One caveat: these are only for endpoint imaging, as any nucleic acid stain like these will, by binding the DNA (nuclear and mitochondrial), will affect DNA function, and thus will inhibit normal transcription and proliferation and eventual affect cell health, and these dyes will come off over time, since they do not bind covalently.
Hi Philip,
I recommend Hoescht 33342 as well. It gets a bit toxic after 2h. I have also used NuclearID from Enzo Sciences which is far red. It works nicely but not efficiently as Hoescht.
With acridine orange you may maintain cellular viability a bit longer than the other dyes.
I can definitely recommend SiR-DNA; it stains in the far red so you can still use GFP tagging for other components and it works well with long-term culture (it bleaches a bit after 10+ hours, but you can reapply it with fresh medium)
I would suggest transfection with some histon - fluorescent tag plasmid. It will take longer than normal staining but phototoxicity is very low and live cell imaging can be done for hours.
I recommend NucLight labelling proteins - these are shown to be non perturbing to cells over long periods of time, and easy to transfect
More details here
http://www.essenbioscience.com/en/products/reagents-consumables/nuclight-red-lentivirus-reagent-ef1a-puro-02ml-cat-no-4625/
best of luck!
There are many nuclear stains but the two best dye versions (and easiest, no transfections required) are Hoechst and SIR-DNA (from Cytoskeleton). Both are bright dyes that when used at the appropriate dose (I use 1ug/ml for Hoechst and 250nM for SIR-DNA) you can timelapse image for 24 hours or more without significant photobleaching. Neither dye (at low doses) inhibits cell division or cell migration. Both are great for cell tracking. Hoechst excites in the 365nm range while SIR-DNA excites with 642nm.
SiR-DNA from Spirochrome would be my suggestion. You have to work out the concentration and timing for the dye to stain the nucleus well but it's nice since it's far red and you can still use GFP channel.
I used SiR-DNA for staining living U2OS cells after they were synchronized using Thymidine. I could observe a release of the SiR-DNA dye after around 1h into vesicles in the cytoplasm... were the cells infected by bacteria, or were they just stressed and have thrown the dye out of the nucleus by themselves?
An improved version of SiR-DNA with up to 10-fold brighter nuclear staining has been recently introduced to the community of biologists and biochemists.
As well, additional colors of DNA stains have been also added (TMR, 580CP, 610CP dyes). All Hoechst-based fluorescent probes are cell-permeable and compatible with live-cell super-resolution microscopy. Samples available upon request from the corresponding author of the paper.
Article Rhodamine-Hoechst positional isomers for highly efficient st...
There are also some commercially available stains from Biotium, Merck and Enzo Life Sciences companies:
NucSpot® Live Cell Nuclear Stains
https://biotium.com/product/nucspot-live-cell-nuclear-stains/
NUCLEAR-ID® Red DNA Stain
http://www.enzolifesciences.com/ENZ-52406/nuclear-id-red-dna-stain/
BioTracker 650 Red Nuclear Dye
http://www.merckmillipore.com/DE/de/product/BioTracker-650-Red-Nuclear-Dye,MM_NF-SCT119?bd=1
FYI Hoechst stains ALWAYS causes cell death in our live cells during time-lapse studies. Yes, we did all the proper controls to make sure it was the dye and not another reason.
Depends on the color. In my experience, the best are: Hoechst dyes in blue, SYTO 9 in green, NucRed Live 647 in the far red. But any nucleic acid binding dye will affect DNA function and be toxic to cells, so they should only be used for end point analysis. If you are wanting to do timelapse imaging or culturing after labeling, then, yes, I would recommend tranfecting for fluorescent protein expression in nuclei, such as with CellLight nuclear GFP or RFP reagents (which are transient transfection and have a very high efficiency for non-hematopoietic mammalian cell lines.
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