At 15:57 26/01/99 -0500, Keith Bahjat wrote: (In response to Joan's question about PI fading) >This message brings up a point that I'd like to see addressed by the experts. Remember the definition: "X" is the unknown quantity, and "spurt" is a drip under pressure. ;~) I am not claiming expert status, but I do have some observations from the operator's vantage point... We often see PerCP fade significantly over an hour or two in room light, the change was enough to ruin an experiment. I have seen tandem conjugates separate into mixtures of tandem plus separate fluorochromes. This separation was variable from tube to tube, and made compensation unworkable! I can't comment on the behaviour of PI in this context, though I don't remember such effects when I was reading lots of DNA samples years ago. I've not seen significant fading of FITC or PE, but I haven't looked very hard; I've always presumed they are less prone to fading. There are many variables in this can of worms, most of which I have no idea of. Most people here take the relatively easy precautions like keeping things covered (most of the time) and on ice unless room temperature (or higher) is specified. Joseph. >In my hands, FITC (as well as PE, PerCP, APC, PI, 7-AAD, and ToPro-3) is very >stable, and no precautions need to be taken while staining or running samples, >as no degradation has ever been seen. >Every clinical and research lab I've been >in stains samples on the bench top and takes no special precautions to avoid >any forms of light. But some researchers here do things like staining in the >dark, encasing samples in aluminum foil, and using red filters on the room >lights in the flow lab for fear of fluorochrome degradation. Based on my >experience, I'm not sure why they fear the light to this extent. I've seen this >happen using the fluorescent scope, but I think those are usually fluors like >Cy2 and Cy3. Are some derivations of fluorescein more photolabile than others, >or am I ruining my samples. > >thanks for any news and views. > >Keith Bahjat >kbahjat@ufl.edu -- Joseph Webster Flow Cytometry Facility Centenary Institute
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