Yeast Cell Cycle by Flow Cytometry

Tuesday, November 18, 2003

Description
Yeast Cell Cycle by Flow Cytometry

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

Vortex tube while adding 1.0 ml cold 70% EtOH.
Store at 4 C (cells keep ~indefinitely).

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.

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.

For staining:

Propidium Iodide
Add 0.5 ml 50 mM Na citrate containing 8 g/ml PI, so that final concentration in the sample is 4 g/ml. There can be non-specific staining of yeast (pombe) ends at higher concentrations if cells are starved, or spores. Cells can be processed immediately or conveniently stored overnight at 4C in the dark before processing the next day. If necessary cells can be stored at this stage for a maximum of a week (4C in the dark). Check them under the fluoresence microscope (red channel) to verify staining.

Sytox Green
Alternatively, add 0.5 ml 50 mM Na citrate containing 2 M Sytox Green, so that final concentration in the sample is 1 M.

(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.

Approximate settings on the FACScan for Propidium Iodide
Detector FSC E00 Gain:3
Detector FL2-A Voltage:890 Gain:2

Approximate settings on the FACScan for Sytox Green
Detector FSC E00 Gain:2
Detector FL1-A Voltage:400 Gain:4

Recipes
Cold absolute ethanol.
0.5 M Na citrate stock (filtered), 50mM diluted stock.
10 mg/ml RNase A (Boil 10 mins, cool, filter and store at -20C).
4 mg/ml Propidium iodide (PI) (filter and store in dark at -20C).
OR

SYTOX Green (Molecular Probes catalog S-7020; 5mM stock in DMSO and stored in dark at -20C)

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Yeast Cell Cycle by Flow Cytometry