I have a client that is staining yeast with PI and then doing cell cycle analysis. From what I understand, the wild type yeast normally spend most of their time in a 2N ploidy state. Upon starvation they arrest in a 1N state. We are trying to determine what different treatments do to the the ratio of the 2 ploidy states. So, we should get a distribution that resembles a G1/G2 that you would see in other eukaryotic sysytems. The problem is that we get a lot of variation in the fluorescence signals. If I set the 2N peak of the wild type at a mean of 400, the 1N peak should be at 200, no? The peaks just seem to migrate all over the scale. The "1N" peaks of some samples are off scale at the bottom and the "2N" peaks of other samples nearly go off the top end. To make matters worse, all the samples on the whole are dim compared to say, calf thymocyte nuclei. I'm running the samples on a FACSCalibur displaying FL2-Area. The calf thymocyte nuclei give a mean of 200 when the voltage on FL2 is between 450 and 500. I have to run at over 900 volts to get the yeast 2N peak at a similar value. Is this normal? Could it be that we're just measuring how well she's permeabilizing the yeast instead of DNA content? (The 1N yeast are supposed to be more resistant to environmental changes) In addition, her samples greatly vary in cell number per tube (somewhere between 10 and 100 fold). How much will this affect the fluorescence intensity detected? She also informed me that this particular yeast elongates and becomes rodshaped when it grows. Could this have an effect? I'd appreciate any input. And this experiment is just the yeast of my worries.... David McFarland Howard Hughes Medical Institute Flow Cytometry Facility Vanderbilt University Medical Center
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