FM4-64 Labeling of Yeast Cells: Difference between revisions
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==Materials== | ==Materials== | ||
*Instead of FM4-64 we use SynaptoRed (Sigma #S6689) | |||
*Media (typically [[YPDA Media]]) | |||
*Dye solution. For each cell line need 250 uL media + 3 uL dye. | |||
==Protocol== | ==Protocol== | ||
#Grow and overnight culture of yeast in the appropriate media. | #Grow and overnight culture of yeast in 4 mL of the appropriate media. | ||
#Spin cells down and resuspend in FM4-64 for 1h. | #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 | #Mount cells on coverslip and analyze by fluorescent microscopy | ||
[[Category:Yeast]] | [[Category:Yeast]] | ||
[[Category:Immunoflouresence]] | [[Category:Immunoflouresence]] | ||
Revision as of 16:47, 7 December 2009
Materials
- Instead of FM4-64 we use SynaptoRed (Sigma #S6689)
- Media (typically YPDA Media)
- Dye solution. For each cell line need 250 uL media + 3 uL dye.
Protocol
- 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