Diane, One normally usually uses FL2 to measure the PI signal (the PE channel - 585/42BP), but many yeast folks use FL3. This is because there is a lot less PI signal in FL2 than in FL3 on *most* cytometers, allowing for more high voltage and better linearity for the DNA measurement. With yeast, the smaller DNA signal makes it worth your while to check both FL2 and FL3 in your system to determine the best channel to use. At Scripps, most yeast PI signal is measured in FL3 on the FACScans (650nm LP), and in FL2 on a FACS Calibur (FL3 is a 670nm LP on the new systems). Since you're measuring nuclear fluorescence, and the geometry of the cells may be unusual depending on the yeast you are assaying, simple pulse height may produce better results than pulse area. This is almost never true for mammalian cells, but often true for yeast. Use an amplifier gain that is low and as much high voltage as reasonable for the best signals. In addition, match the sample and sheath buffers. Some yeast protocols use citrate buffers that have a different refractive index than PBS, producing optical artifacts you don't need. Some of our yeast folks bring their own sheath buffer, and some use PBS sample buffer to accomplish this. Good luck, Joe Joseph Trotter, Director Flow Cytometry Core Facility The Scripps Research Institute 10550 North Torrey Pines Road La Jolla, CA 92037 On Tue, 18 Jan 2000, Diane M Sharp wrote: > Hello all, > > I have recently been asked to run cell cycle on yeast. I have run endless > samples for cell cycle on tumor lines, but never on yeast. I will be > using propidium iodide. Is this a difficult assay? I would appreciate > any comments from folks who have already run this type of sample. > Thanks in advance for your help! > > Diane Sharp > DuPont Pharmaceuticals > Glenolden, PA >
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