>The question has come up again - Can one sort chromosomes with low-power >Argon laser(s) >(ONE water cooled & tunable, one 488 nm ONLY) in a FACStar+? > >1) single laser 488 nm TOTO-1 (AT selective) and 7-AAD (GC selective)? > I spent several years evaluating a whole lot of combinations theoretically usable with 488 nm, including that one, and, under the best of circumstances all you see with any of them is a hint that some groups of chromosomes aren't on the 45 degree line. The CV's of the individual clusters aren't as good as you would get with propidium alone. 7-AAD gives a very low intensity signal, which is part of the problem with CV's. >2) single laser UV (350/360 nm)? I don't know whether anybody has worked up potential bivariate dye combinations for single-beam UV excitation; most folks who can afford the UV can afford a 457 (or a 441 He-Cd). > >3) 457 nm and 488 nm dual lasers? > Mithramycin/ethidium is a popular DNA stain for excitation by arc lamp systems (436 nm); a mithramycin or chromomycin combination with ethidium or propidium should give you some separation in a bivariate space, although I don't know of any reports on the subject. >4) 350/360 nm and 488 nm dual lasers? > The Los Alamos group has reported on this in Cytometry some years back, using Hoechst/TOTO or YOYO; that would probably be your best bet given the available lasers. I got some fair bivariate histograms using TOTO-1 and TOTO-3 with 488 and 633 nm excitation; Tom Frey at B-D did even better, using thiazole orange and thiazole blue, or maybe it was TO-PRO-1 and TO-PRO-3, according to a poster he presented at the 1993 ISAC Meeting. As I recall, this stuff didn't find its way into his published paper in Cytometry, which dealt with using low power UV and 441 from He-Cd lasers for bivariate chromosome sorting with Hoechst/chromomycin. I have played that game, so has Kit Snow at Coulter, in conjunction with Scott Cram. A FACStar is probably going to want 100 mW or so of UV and blue for bivariate analysis; if you can get UV from the tunable argon, you might consider getting a 100 mW 441 nm He-Cd, which is probably available for under $20,000, because that should do OK for chromomycin (if the laser isn't too noisy). -Howard
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