906
edits
Changes
updated synaptored concentration
==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.#Quench Spin down cells for and wash 2x with media.#Add cells to 5 mL media and mix. Take a 1 mL sample and measure A600#Incubate 3hwith 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
[[Category:Yeast]]
[[Category:Immunoflouresence]]
see http://www.mcdb.lsa.umich.edu/labs/klionsky/FM4-64labeling.pdf
