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Yeast Cell Cycle

Sytox Green is better than PI.

Materials

RNAse 2 mg/ml RNAse A in 50 mM Tris pH 8.0 & 15 mM NaCl (boil for 5 -10 minutes and allow to cool, aliquot and freeze)	Protease 5 mg/ml pepsin, 4.5 microliter/ml concentrated HCl in H2O
SYTOX Green Staining Solution 1 mM SYTOX Green in 50 mM Tris pH 7.5

Method

1. Pellet 10⁷ cells and re-suspend in 1.5 ml H₂O.
2. Slowly add 3.5 ml 100% EtOH. Fix at least 1 hr RT (or overnight in the refrigerator).
3. Wash fixed cells once with 1.0 ml H₂O.
4. Re-suspend in 0.5 ml RNAse solution and incubate for 2 - 12 hours at 37º C.
5. Pellet and re-suspend in protease solution and incubate 20 minutes at 37º C.
6. Pellet and re-suspend in 0.5 ml 50 mM Tris pH 7.5.
7. Can be stored at 4º C.
8. Add 50 - 100 microliters of processed cells to 500 - 900 microliters of SYTOX Green Staining Solution in Falcon® 12 X 75 polystyrene tubes [35]2052, 2054, or 2058.(DO NOT SUBSTITUTE TUBES.)
9. Sonicate and analyze.

  Reference
Haase SB, Reed SI. Improved flow cytometric analysis of the budding yeast cell cycle. Cell Cycle. 2002 Mar-Apr;1(2):132-6. PMID: 12429922

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Dennis J. Young, UCSD Cancer Center Flow Cytometry Core Facility