Preparation of Yeast Cells for Flow Cytometry

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Tony Rook
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Preparation of Yeast Cells for Flow Cytometry

Please find the following protocol:

Preparation of Yeast Cells for Flow Cytometry

Procedure:

1. Grow an overnight culture of the desired yeast strain in YPD.

2. Dilute the culture into YPD until the optical density is 0.05 at 600 nm (OD600 = 0.05).

3. Grow cells to OD600 = 0.5. For publication quality or to ensure that cells are not synchronized and are growing logarithmically, dilute cells back to OD600 = 0.1 and grow to OD600 = 0.4 to 0.6.

4. Pellet cells from 1 ml of culture by centrifugation in a microcentrifuge at full speed for 5 to 10 sec.

5. Remove the supernatant and resuspend the cells in 250 μl TE.

6. Add 750 μl of 100% Ethanol and incubate for 1 hr at room temperature.

7. Pellet the cells in a microcentrifuge. Cells are more difficult to pellet after ethanol fixation.

8. Remove the supernatant and resuspend the cells in TE (You can store the cells at this point in TE/Azide at 4°C.), repellet the cells and resuspend in TE.

9. Repellet the cells.

10. Resuspend the cells in TE/RNase and incubate at 37°C for 4 hr.

11. Pellet the cells.

12. Remove the supernatant and resuspend the cells in PBS and pellet cells again.

13. Resuspend the cells in PI/PBS.

14. Incubate the cells overnight at 4°C.

15. Resuspend the cells by a quick vortexing and dilute them 10-fold in PBS.

16. Sonicate the cells for 5 sec at a medium-low power setting.

17. Proceed with flow cytometry of yeast cells.

Solutions:

PI/PBS
50 μg/ml Propidium Iodide in PBS

TE/RNase
20 mM EDTA
0.1% (w/v) RNase
0.2 M Tris, pH 7.5

TE/Azide
20 mM EDTA
0.01% (w/v) Sodium Azide (CAUTION Biohazard!)
0.2 M Tris, pH 7.5

TE
20 mM EDTA
0.2 M Tris, pH 7.5

YPD
Autoclave 20 min, cool to room temperature
10 g/liter Yeast Extract
20 g/liter Peptone
20 g/liter d-Glucose

Bioreagents and Chemicals:

Tris
Peptone
RNase
Ethanol
Sodium Azide
D-Glucose
Propidium Iodide
EDTA
Yeast Extract

Reference Link:

http://www.bio.com/protocolstools/protocol.jhtml?id=p821