FM4-64 Labeling of Yeast Cells: Difference between revisions

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==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 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.

#~~Quench~~ cells ~~for~~ 3h.

#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

[[Category:Yeast]]

[[Category:Immunoflouresence]]

see http://www.mcdb.lsa.umich.edu/labs/klionsky/FM4-64labeling.pdf

@@ Line 1: / Line 1: @@
 ==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 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.
-#Quench cells for 3h.
+#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
 [[Category:Yeast]]
 [[Category:Immunoflouresence]]
+see http://www.mcdb.lsa.umich.edu/labs/klionsky/FM4-64labeling.pdf