Fig. 6. Both monomethyl and dimethyl M-C₆-NBD-PE are trafficked to the vacuole in yeast. The wild-type diploid strain CRY3 was grown to early log phase in SDC at 30°C. Cells were then labeled with 5 µM DMSO-solubilized lipid for 45 minutes at 30°C. After being washed twice with room temperature SDC, DAPI was added to label nuclear and mitochondrial DNA. Cells were incubated at 30°C for 15 additional minutes, harvested and washed twice with ice-cold SCNaN₃ prior to analysis and imaging by fluorescence microscopy.