FM4-64 Labeling of Yeast Cells - Revision history

Davebrid: updated synaptored concentration

2010-01-27T15:24:16Z

updated synaptored concentration

← Older revision		Revision as of 15:24, 27 January 2010
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	==Materials==		==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'''		*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]])		*Media (see [[YPD Media and Agar]])
	*Dye solution. For each cell line need ~~190~~ uL media + 60 uL dye.		*Dye solution. For each cell line need 200 uL media + 1 uL dye.

	==Protocol==		==Protocol==

Davebrid: Changed dye concentration to match klionsky protocol

2010-01-20T16:26:38Z

Changed dye concentration to match klionsky protocol

← Older revision		Revision as of 16:26, 20 January 2010
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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 ~~250~~ uL media + 3 uL dye.		*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

Davebrid at 19:48, 7 December 2009

2009-12-07T19:48:03Z

← Older revision		Revision as of 19:48, 7 December 2009
Line 1:		Line 1:
	==Materials==		==Materials==
	*Instead of FM4-64 we use SynaptoRed (Sigma #S6689). This is resuspended 1mg to 500 uL (2ug/mL) with DMSO.		*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 250 uL media + 3 uL dye.		*Dye solution. For each cell line need 250 uL media + 3 uL dye.

Davebrid: added SynaptoRed concentration

2009-12-07T19:40:25Z

added SynaptoRed concentration

← Older revision		Revision as of 19:40, 7 December 2009
Line 1:		Line 1:
	==Materials==		==Materials==
	*Instead of FM4-64 we use SynaptoRed (Sigma #S6689)		*Instead of FM4-64 we use SynaptoRed (Sigma #S6689). This is resuspended 1mg to 500 uL (2ug/mL) with DMSO.
	*Media (see [[YPD Media and Agar]])		*Media (see [[YPD Media and Agar]])
	*Dye solution. For each cell line need 250 uL media + 3 uL dye.		*Dye solution. For each cell line need 250 uL media + 3 uL dye.

Davebrid at 16:47, 7 December 2009

2009-12-07T16:47:57Z

← Older revision		Revision as of 16:47, 7 December 2009
Line 1:		Line 1:
	==Materials==		==Materials==
	*Instead of FM4-64 we use SynaptoRed (Sigma #S6689)		*Instead of FM4-64 we use SynaptoRed (Sigma #S6689)
	*Media (~~typically~~ [[~~YPDA~~ Media]])		*Media (see [[YPD Media and Agar]])
	*Dye solution. For each cell line need 250 uL media + 3 uL dye.		*Dye solution. For each cell line need 250 uL media + 3 uL dye.

Davebrid: added details from Emily

2009-12-07T16:47:33Z

added details from Emily

← Older revision		Revision as of 16:47, 7 December 2009
Line 1:		Line 1:
	==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.
	#~~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		#Mount cells on coverslip and analyze by fluorescent microscopy

	[[Category:Yeast]]		[[Category:Yeast]]
	[[Category:Immunoflouresence]]		[[Category:Immunoflouresence]]

Davebrid: initial page

2009-10-05T19:57:19Z

initial page

New page

==Materials==

==Protocol==
#Grow and overnight culture of yeast in the appropriate media.
#Spin cells down and resuspend in FM4-64 for 1h.
#Quench cells for 3h.
#Mount cells on coverslip and analyze by fluorescent microscopy

[[Category:Yeast]]
[[Category:Immunoflouresence]]