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Yeast Cell Cycle Flow Cytometry Protocol

Yeast Cell Cycle by Flow Cytometry



Susan Forsburg's method for Schizosaccharomyces pombe

Reagents

Protocol

  1. Spin down 107 cells from an exponentially growing culture - 2000 rpm for 5 mins. Pour off supernatant.

  2. Vortex tube while adding 1.0 ml cold 70% EtOH.

  3. When you want to process the cells, take 0.3 ml (this will be 2-3 x 106 cells, assuming a little loss in the washing) and add to 3 ml 50 mM Na citrate in a 5ml Falcon tube. Mix and spin 2000 rpm for 5 mins.

  4. Discard supernatant and resuspend pellet in 0.5 ml 50mM Na citrate containing 0.1 mg/ml RNase A. Leave in 5 ml Falcon tube and put in 37 °C room for 2 h.

  5. For staining:

  6. (Optional) Just before processing the cells, sonicate for 45 s again leaving cells in the 5 ml Falcon tubes. Sonication prevents doublets of cells which give spurious peaks and is particularly useful if your cells have varying DNA contents and will clean up spores or wee mutants.

  7. Approximate settings on the FACScan for Propidium Iodide

  8. Approximate settings on the FACScan for Sytox Green

Points to bear in mind

General reference (PI method) : Sazer and Sherwood (1990) J. Cell Sci 97:509-516


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