From: Robert C. Leif (rleif@rleif.com)
Date: Wed Aug 20 2003 - 01:37:11 EST
Mario et al. I said the Quantum Dots half life does NOT preclude their use in flow; while, the long half life of the Quantum Dyes(R) does preclude flow. Since multiple antibodies can bind to a myriad of sites on a Quantum Dot, obtaining a one to one conjugate would be difficult. However, I agree that one can live with complex mixtures. However, I try to avoid them because their use can increase the complexity of the preparation and the experiment. Bob Leif Robert C. Leif, Ph.D. Email rleif@rleif.com -----Original Message----- From: Mario Roederer [mailto:roederer@drmr.com] Sent: Monday, August 18, 2003 6:37 PM To: cyto-inbox Subject: RE: [Quantum Dots] Quantum dots can indeed be used to label antibodies, although the chemistry for doing so is still nascent and needs optimization. I would strongly warn against trying this with currently-available products; while some companies claim they have products that they can conjugate to antibodies (which is true), the resulting product is likely to be worthless--the nonspecific binding of the dots is huge, rendering any specificity zero. However, I am optimistic that this problem will be overcome soon. In the meantime, you can use the streptavidin conjugates. In terms of brightness, the dots vary significantly by which you use. In general, the further red, the brighter (as expected based on the larger size and therefore large absorbance, plus other factors). The fact that they don't bleach is not really an advantage for flow cytometry, where bleaching is rarely an issue with the extremely short excitation time (microseconds). Their lack of bleaching makes for a huge advantage in microscopy, I would guess. The emission spectra are not as narrow as I would have hoped, nonetheless, they are narrow enough that theoretically one could detect many simultaneously off one laser with low cross-over. And while, as Bill Telford notes, there is reasonable cross-over into other lasers, it is not insurmountable--it isn't close to as bad as Cy5PE into APC, for example, and some dots have very little spectral overlap into current channels. One real advantage is that nothing currently used spills over into the quantum dot channels. Bob Leif suggests that their half-lives preclude use in flow cytometry, but this is certainly not the case; for example, we find the 655 quantum dot to be as bright as almost any color we use (e.g., APC-like!). Given that, I find it difficult to believe that their half life is in the millisecond range. Also, Bob notes that they have no specific attachment points for chemistry, but this makes them no different than PE or APC (which also have no specific attachment points); it's relatively trivial to adjust the chemistry to get a 1:1 dot:antibody conjugation. If you're in competition with them, Bob, then ... well, all I can say is, I'm sorry :( I think Quantum dots will, in the next few years, play a fairly significant role in flow cytometry, as yet another series of colors in the expanding fluorescence arsenal. I don't think that they will be commonly-used on "yesterday's" flow cytometers, as they make most sense when using UV (or near-UV) excitation. However, I do believe that they will become a common tool in the realm of 6+ color flow cytometry within a few years. mr (Yup, I too have no commercial ties to any company involved in this technology. I do kick myself on a daily basis for not having thought of it first, though.)
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