Measuring Lipid Droplet Size in Yeast

To analyze the size and number of LDs, yeast cells were grown in YSC medium supplemented with 2% (w/v) glucose. After growing for 24 h at 30 °C with shaking at 250 rpm, the cells were washed twice with phosphate-buffered saline (PBS) and fixed with 3.7% formaldehyde in PBS as described previously [11 (link), 49 (link), 50 (link)]. Next, the cells were stained with 5 µM BODIPY 493/503 dye for 30 min at 30 °C. Then, the cells were washed twice with PBS and observed with a Zeiss-LSM 780 multiphoton confocal microscope (Zeiss, AG, Germany) equipped with a Plan-Apochromat 63x/1.4 NA oil immersion objective. Confocal images were analyzed using ImageJ software (National Institutes of Health, Bethesda, USA) and ZEN imaging software (Zeiss, AG, Germany).