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Is Draq7TM capable of distiguishing diploid from tertraploid and octoploid nuclei by FACS? We are currently using propidium iodide, which works well but overlaps with the emission of our Tomato Red co-stain. We do not permeabilize the nuclei but have found that antibodies cross the nuclear membrane, so we are assuming this will too.
Asked on Aug 24 2012
Thank you for your enquiry.
We recommend Draq5TM, ab108410, for your application, rather than Draq7TM. The laboratory has provided the following notes which describe the differences between the two. In brief, the max emission wavelengths are the same, about 665nm, compared to Tomato Red at about 585nm and propidium iodide at 617nm. It appears that there is some overlap between the DRAQ and Tomato Red emission spectra, but it should be possible to gate out the overlap in the cytometer software.
Click here (or use the following: https://www.abcam.com/index.html?datasheet=108410).
From the lab:
"We would still recommend the use of DRAQ5 rather than DRAQ7, although you are almost certainly right in your assumption of these nuclei being permeant to DRAQ7 (as they are to PI). However, the issue will be the resolution that can be achieved in the DNA profiles, since this is what you aim to gain by preparing nuclei (Vindelov method) rather than using intact cells, though the separation required here is much less than that for hypoploidy, for example. The likelihood is that DRAQ5 will give better CV's than DRAQ7 and, perhaps, more importantly has a pedigree for DNA content analysis of many papers in the last 10+ years. DRAQ5 can be used to measure DNA content in intact, unfixed cells in complex mixtures such as bone marrow, blood and so will work independent of the permeability of the membranes and as such allows a panoply of control experiments. In spectral terms, DRAQ5 and DRAQ7 are identical."
Please let me know if you have any questions. I will be out next week but one of my colleagues will be able to help.
Answered on Aug 24 2012