Ray Hester writes- >I don't remember the following having been discussed (but then my memory is >not that great!): > >For years I've noticed when we run propidium iodide-stained samples for >ploidy analysis that the PI fluorescence (488 Ex, 585/42 EM) gradually >increases for the first 10 or 15 secs and then finally stabilizes (samples >are in the staining buffer with PI; sheath fluid is PBS). > >Several years ago I was at a _major_ manufacturer's instrument training >session and they were touting the fact that analyses on their instruments >were not susceptible to this gradual increase because of their (proprietary) >sample tubing and sure enough we ran PI stained samples (I can't remember >exactly what they were) and plotted fluorescence vs time, there was no >variation in fluorescence intensity. > >One can wait until the fluorescence seems to stabilize so that CVs are not >affected but it would be nice to know on the front end that this phenomenon >is not a potential factor. > >Any suggestions about tubing and/or sheath fluid to prevent the above would >be appreciated. PI fluorescence intensity is sensitive to ionic strength of the solution; mixing of the hypotonic staining buffer with a saline sheath (rather than effects of sample tubing) is generally thought to be responsible for the shifting fluorescence. If the sheath and sample have the same ionic strength, fluorescence shifts should be minimal. -Howard
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