• Complete+Jablonski+Diagram
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« Your first LSRII experiment - PMT Optimization | Main | Using FlowJo to calculate compensation »

April 27, 2006


John Altman


The Galahad worksheet requires at least five small revisions, and maybe some clarification.

1) The bandpass filter on the "B" detector on the red laser should be changed.

2) The alternate label on this same channel is not Alexa 610, but Alexa 680. The antibody suppliers seem to have settled on Alexa 700—and for the most part, we have too, with one exception. We can make Alexa680 tetramers, but we can't make Alexa700 tetramers. So, you might sometimes see Alexa680. Otherwise, steer people away from it.

3) On the "C" detector on the violet laser, you should move QD565 to the top of the list, and delete the other two, which are better for the "D" detector.

4) Pac Orange should probably be listed under the D detector on the violet laser. I don't know which of these labels should assume the coveted spot at the head of the list.

5) The "B" detector on the blue laser is obviously complicated. In the first column, you've listed PerCP-Cy5, which I doubt anyone as ever made. You almost certainly mean PerCP.

The emission spectra for the DNA dyes — EMA, 7-AAD, and PI—are very broad, and they can be detected in lots of channels (see attached figure -- I've used ethidium homodimer in place of EMA -- I don't know if it's a good match but it was the best I could do), so users have choices. The good thing about them is that since they are used to exclude cells —dead ones—you don't have to worry too much about their spillover into other channels, since there will be little-to-no contribution to the signals on the live cells. You should probably still set up a comp tube for them, but it's a bit tricky. I'm not sure you have to change anything here, just be aware of it. Since so many of us here are working with fixed samples, we probably don't have as many people using these dyes as you might think.

Finally, it is marginally noteworthy that the red laser on Gal is shifted by probably 5 nm relative to Guin. When you look at the excitation spectra, you see that as you red-shift the laser line, you are climbing up the excitation curves of both APC and Alexa700. This means better excitation, which means brighter signals. How much better? Hell if I know. Do our users need to know it? Probably not. But if we ever get a green laser, or one of the 568 jobs that Telford has published on, we'll use arguments like this to justify the purchase.

Suzanne Mertens

Thanks for the critique. I've anwered your questiosn by revisiting the BD spectra viewer and making appropriate edits to the vairous panle worksheets. Links to updates will be made avaialbel in the next blog posting.

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