FM4-64 Labeling of Yeast Cells: Difference between revisions
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Changed dye concentration to match klionsky protocol |
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*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''' | *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]]) | *Media (see [[YPD Media and Agar]]) | ||
*Dye solution. For each cell line need | *Dye solution. For each cell line need 190 uL media + 60 uL dye. | ||
==Protocol== | ==Protocol== | ||
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[[Category:Yeast]] | [[Category:Yeast]] | ||
[[Category:Immunoflouresence]] | [[Category:Immunoflouresence]] | ||
see http://www.mcdb.lsa.umich.edu/labs/klionsky/FM4-64labeling.pdf | |||
Revision as of 16:26, 20 January 2010
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
- 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