Difference between revisions of "FM4-64 Labeling of Yeast Cells"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (initial page) |
Davebridges (Talk | contribs) (updated synaptored concentration) |
||
| (5 intermediate revisions by the same user not shown) | |||
| Line 1: | Line 1: | ||
==Materials== | ==Materials== | ||
| + | *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. | ||
==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]] | ||
| + | |||
| + | see http://www.mcdb.lsa.umich.edu/labs/klionsky/FM4-64labeling.pdf | ||
Latest revision as of 15:24, 27 January 2010
Materials
- 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.
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
see http://www.mcdb.lsa.umich.edu/labs/klionsky/FM4-64labeling.pdf
