I posted a question a while ago about the ins and outs of DNA analysis on yeast. I received numerous informative responses. Thanks. Information to know: 1) Yeast have ~300X less chromosomal DNA than mammalian cells. This explains what I considered to be a very high voltage needed to place the yeast DNA peak at a similar spot on the scale vs calf thymocytes. 2) Yeast have a much larger ratio of mitochondrial to chromosomal DNA. First of all, this means that it is more difficult to accurately do cell cycle analysis. This is exacerbated by the fact that mitochondrail number can vary greatly depending on culture conditions. This becomes more pronounced when using AT-specific stains like Hoechst and DAPI since the mitochondrial DNA is about 80% AT. 3) Permeabilization is very important and must be consistent or you may see great differences in staining/fluorescence intensity. (I think this is my main problem, although I haven't confirmed it yet, especially with yeast that have been starved and arrested in a G1 cyst-like state, making them very resistant to environmental changes.) 4) Cell number/dye concentration also needs to be consistent to get consistent staining. 5) Multinucleation and formation of germ tubes can cause consistency problems as well as difficulty with doublet discrimination, clumping, turbulence, etc. Some of this info was taken directly from responses on this forum and also from the following sources I was referred to: Lynch ME, KuKuruga MA, Nakeff A, Fidel PL, Sobel JD. Flow cytometric analysis of germ tube formation in Candida albicans. J Med & Vet Mycology 31: 367-376, 1993. Dien BS, Peterson MS, Srienc F. Cell-cycle analysis of Saccharomyces cerevisiae. Methods in Cell Biology 42: 457-475, 1994. That is all. David McFarland Howard Hughes Medical Institute Flow Cytometry Facility Vanderbilt University Medical Center
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