FM4-64 Labeling of Yeast Cells

Materials

• Instead of FM4-64 we use SynaptoRed (Sigma #S6689). This is resuspended 1mg to 500 uL (2ug/mL) with DMSO and stored in Molecular Biology Stuff
• Media (see YPD Media and Agar)
• Dye solution. For each cell line need 190 uL media + 60 uL dye.

Protocol

1. Grow and overnight culture of yeast in 4 mL of the appropriate media.
2. Spin cells down and resuspend in dye solution FM4-64. Wrap in alluminum foil and incubate with shaking at 24C for 1h.
3. Spin down cells and wash 2x with media.
4. Add cells to 5 mL media and mix. Take a 1 mL sample and measure A600
5. Incubate 3h with shaking at 24C. Take a 1 mL sample and measure A600. This should be one full doubling from the previous reading.
6. Mount cells on coverslip and analyze by fluorescent microscopy

see http://www.mcdb.lsa.umich.edu/labs/klionsky/FM4-64labeling.pdf