FM4-64 Staining in Yeast Cells

FM4-64 staining with small modifications was performed as described [52 (link)]. Yeast cells were diluted to an OD₆₀₀ = 0.1 and were grown for 12 h in liquid SC-medium. An aliquot (2 mL) was washed once in YEPD and resuspended in 100 µL YEPD supplemented with 20 µM FM4-64. After a 20 min incubation at 28 °C, cells were wash with SC-medium and incubated for 1 h in 100 µL liquid SC medium. Moreover, 50 µL of this cell suspension was used as a control, 50 µL was supplemented with 1 M NaCl and incubated for >30 min. Cells stained by FM4-64 were analyzed with a × 100 objective (NA = 1.4) using either a Carl Zeiss AG Axioscope (Oberkochen, Germany) or a Nikon (Tokyo, Japan) Eclipse Ni-U equipped with a DS-Fi2 digital camera.