Hi Adrian I use Coulter Flowcheck beads for all the 488 channels, they have good cvs and fluoresce over a pretty broad range. I routinely use fitc, pe, pe/cy5 and pe/cy7. If the alignment is OK for FL1 and FL2 (OK the 530/40 and 575/40 or whatever) then the other two will probably be OK, you may have to tweak the two dichroics a bit. I use Molecular Probes 6u 633 beads for the 633 laser channels, which are set up for APC and APC/cy7, incidentally I use the same beads for the time delay calibration in the Caliburs. Both these beads give some signal in the UV channels but if you want good cvs you might want to use special UV beads like the Molecular Probes 2.5u UV beads. Using 6 colrs with pe/cy7 and apc/cy7 is really not much harder than using 4 colors but dont let anyone know that. Doing compensation right is pretty key. These two dyes are not that bright so are best used for bright markers. I've heard good things about the Alexa dyes but dont use them myself. My uv laser is an I90 Krypton so I can switch to violet and do CFP or cascade blue and yellow but no one ever wants to here, we are simple folk up here in the woods. Best of luck Simon >>> Adrian Smith <A.Smith@centenary.usyd.edu.AU> 09/06/02 18:12 PM >>> To all the Vantage users out there (and those doing lots of colours), We are (still) trying to set up our Vantage (DiVA) for >6 colours and we are wondering what people are using for "tweaking" alignments, voltages etc for far red and UV fluorochromes. Last time we checked with BD in Australia they didn't have any beads to recommend or sell us. We have tried with various stained cells but we are looking for something more reproducible for initial setup and optimisaion. We are also interested in getting some idea of the settings people use and the levels we should be expecting from various flurochromes so we have a rough baseline from which to work. At this stage we are completely open to using any combination of fluorochromes - we just want to use more than 4! The Vantage is set up with three lasers (UV, 488, 633) and 8 detectors (2,4,2) or which 3 or 4 are supposed to be the far-red type. Any comments and suggestions would be most welcome! Thanks, Adrian -- ______________________________________________ Adrian Smith (Research Officer) T CELL BIOLOGY GROUP Centenary Institute of Cancer Medicine & Cell Biology Locked Bag No.6 Newtown, NSW 2042 AUSTRALIA. Ph: (02) 9565-6198 Fax: (02)-9565-6103
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