Detecting Apoptosis in Yeast Cells

Exposed phosphatidylserine was detected by reaction with FITC-coupled annexin V (ApoAlert Annexin V Apoptosis Kit; CLONETECH Laboratories, Inc., Palo Alto, CA). Yeast cells were washed in sorbitol buffer (1.2 M sorbitol, 0.5 mM MgCl₂, 35 mM potassium phosphate, pH 6.8), digested with 5.5% glusulase (Boehringer Mannheim) and 15 U/ml lyticase (Sigma Chemical Co.) in sorbitol buffer for 2 h at 28°C, harvested, washed in binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂; CLONETECH Laboratories, Inc.) containing 1.2 M sorbitol buffer, harvested and resuspended in binding buffer/sorbitol. 2 μl annexin-FITC (CLONETECH Laboratories, Inc.) and 2 μl propidium iodide (500 μg/ml) were added to 38 μl cell suspension, and then incubated for 20 min at room temperature. The cells were harvested, suspended in binding buffer/sorbitol, and applied to a microscopic slide.