FM4-64 Labeling of Yeast Cells
From Bridges Lab Protocols
- Instead of FM4-64 we use SynaptoRed (Sigma #S6689). This is resuspended 5mg to 514 uL (16mM) with DMSO and stored in Molecular Biology Stuff
- Media (see YPD Media and Agar)
- Dye solution. For each cell line need 200 uL media + 1 uL dye.
- Grow and overnight culture of yeast in 4 mL of the appropriate media.
- Spin cells down and resuspend in dye solution FM4-64. Wrap in alluminum foil and incubate with shaking at 24C for 1h.
- Spin down cells and wash 2x with media.
- Add cells to 5 mL media and mix. Take a 1 mL sample and measure A600
- Incubate 3h with shaking at 24C. Take a 1 mL sample and measure A600. This should be one full doubling from the previous reading.
- Mount cells on coverslip and analyze by fluorescent microscopy