McClean:Phluorin Calibration - Revision history

Megan N McClean: /* Notes */

2012-06-06T15:54:00Z

Notes

←Older revision		Revision as of 15:54, 6 June 2012
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	Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.		Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
	*[[User:Megan N McClean\|Megan N McClean]] 10:33, 5 June 2012 (EDT) Currently, we assay pHluorin with two separate filter cubes (with different excitation filters, but the same emission filter). Eventually we will move the excitation filters and the emission filter onto the microscope's filter wheels, so that alignment of the filter cubes in the turret is not an issue and the to cut down on the switching time.		*[[User:Megan N McClean\|Megan N McClean]] 10:33, 5 June 2012 (EDT) Currently, we assay pHluorin with two separate filter cubes (with different excitation filters, but the same emission filter). Eventually we will move the excitation filters and the emission filter onto the microscope's filter wheels, so that alignment of the filter cubes in the turret is not an issue and the to cut down on the switching time.
-	*[[User:Megan N McClean\|Megan N McClean]] 11:53, 6 June 2012 (EDT) Image Processing: We should probably add, in the image processing algorithm, ~~and~~ alignment step (Plugins->Registration->StackReg, or similar) between the GFP and the Phluorin images. Thus far, there doesn't seem to be a large shift between the two images, but there does seem to be some and this causes some error in measurement of the ratio.	+	*[[User:Megan N McClean\|Megan N McClean]] 11:53, 6 June 2012 (EDT) Image Processing: We should probably add, in the image processing algorithm, an alignment step (Plugins->Registration->StackReg, or similar) between the GFP and the Phluorin images. Thus far, there doesn't seem to be a large shift between the two images, but there does seem to be some and this causes some error in measurement of the ratio.
	*[[User:Megan N McClean\|Megan N McClean]] 11:29, 6 June 2012 (EDT) When I did an experiment on 6/5/2012, I noticed that after I permeabilized the cells and put them in appropriate pH buffers a large fraction of the cell population (this was with strain yMM1107 Mat alpha HAP1+ hoΔ::natMXAC-TDH3prom-Phluorin) went dark. Could this be because I over permeabilized the cells (incubated for 55minutes with digitonin instead of 30 minutes) or some other factor from the permeabilization and buffer protocol? In any event, this is something to think about optimizing for the future.		*[[User:Megan N McClean\|Megan N McClean]] 11:29, 6 June 2012 (EDT) When I did an experiment on 6/5/2012, I noticed that after I permeabilized the cells and put them in appropriate pH buffers a large fraction of the cell population (this was with strain yMM1107 Mat alpha HAP1+ hoΔ::natMXAC-TDH3prom-Phluorin) went dark. Could this be because I over permeabilized the cells (incubated for 55minutes with digitonin instead of 30 minutes) or some other factor from the permeabilization and buffer protocol? In any event, this is something to think about optimizing for the future.

Megan N McClean: /* Notes */

2012-06-06T15:53:42Z

Notes

←Older revision		Revision as of 15:53, 6 June 2012
Line 142:		Line 142:
	Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.		Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
	*[[User:Megan N McClean\|Megan N McClean]] 10:33, 5 June 2012 (EDT) Currently, we assay pHluorin with two separate filter cubes (with different excitation filters, but the same emission filter). Eventually we will move the excitation filters and the emission filter onto the microscope's filter wheels, so that alignment of the filter cubes in the turret is not an issue and the to cut down on the switching time.		*[[User:Megan N McClean\|Megan N McClean]] 10:33, 5 June 2012 (EDT) Currently, we assay pHluorin with two separate filter cubes (with different excitation filters, but the same emission filter). Eventually we will move the excitation filters and the emission filter onto the microscope's filter wheels, so that alignment of the filter cubes in the turret is not an issue and the to cut down on the switching time.
		+	*[[User:Megan N McClean\|Megan N McClean]] 11:53, 6 June 2012 (EDT) Image Processing: We should probably add, in the image processing algorithm, and alignment step (Plugins->Registration->StackReg, or similar) between the GFP and the Phluorin images. Thus far, there doesn't seem to be a large shift between the two images, but there does seem to be some and this causes some error in measurement of the ratio.
	*[[User:Megan N McClean\|Megan N McClean]] 11:29, 6 June 2012 (EDT) When I did an experiment on 6/5/2012, I noticed that after I permeabilized the cells and put them in appropriate pH buffers a large fraction of the cell population (this was with strain yMM1107 Mat alpha HAP1+ hoΔ::natMXAC-TDH3prom-Phluorin) went dark. Could this be because I over permeabilized the cells (incubated for 55minutes with digitonin instead of 30 minutes) or some other factor from the permeabilization and buffer protocol? In any event, this is something to think about optimizing for the future.		*[[User:Megan N McClean\|Megan N McClean]] 11:29, 6 June 2012 (EDT) When I did an experiment on 6/5/2012, I noticed that after I permeabilized the cells and put them in appropriate pH buffers a large fraction of the cell population (this was with strain yMM1107 Mat alpha HAP1+ hoΔ::natMXAC-TDH3prom-Phluorin) went dark. Could this be because I over permeabilized the cells (incubated for 55minutes with digitonin instead of 30 minutes) or some other factor from the permeabilization and buffer protocol? In any event, this is something to think about optimizing for the future.

Megan N McClean: /* Image Analysis */

2012-06-06T15:52:00Z

Image Analysis

←Older revision		Revision as of 15:52, 6 June 2012
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	Briefly, the procedure is as follows:		Briefly, the procedure is as follows:
	# Open the GFP images in ImageJ as a series (File->Import->Image Sequence)		# Open the GFP images in ImageJ as a series (File->Import->Image Sequence)
-	# B	+	# Background subtract the images (Process->Subtract Background; Usually for yeast keeping the rolling ball radius at 50 works well)
		+	# Convert the image to 32-bit (Image->Type->32-bit)
		+	# Repeat steps 1-3 with the Phluorin cube images
		+	# Threshold each image, so that the background is set to NaNs (Image->Adjust->Threshold; when satisfied with the threshold click 'apply' and when asked to set the background to NaNs choose this option)
		+	# Divide the phluorin images by the GFP images (Process->Image Calculator->Divide)
		+	#* This image now contains all the background pixels set to NaNs and within the cells each pixel represents the ratio of emission at 410 vs 470nm (See Notes below)
		+	# To get the average ratio of individual cells, we need to identify cells. To do this:
		+	#* Re-open the GFP images as a series
		+	#* Threshold them (Image->Adjust->Threshold; Apply)
		+	#* Break apart cell clumps and fill holes (Process->Binary->Watershed; Process->Binary->Fill Holes)
		+	#* Add each cell (each "particle") to the ROI (region-of-interest) manager (Analyze->Analyze Particles; make sure "Add to Manager" is selected and hit "OK")
		+	#* Select all ROI's in the ROI manager, make sure that the ratio images are the active window (click on them), and hit measure. This will produce a results file with all of the measurements you have selected (to change go to Analyze->Set Measurements) for each ROI in each slice (each image in the image series). You can then plot the desired values in your favorite program (Matlab, Excel, etc).

	==References==		==References==

Megan N McClean: /* Image Analysis */

2012-06-06T15:41:14Z

Image Analysis

←Older revision		Revision as of 15:41, 6 June 2012
Line 118:		Line 118:
	===Image Analysis===		===Image Analysis===
	To do a quick analysis of the data, we use ImageJ and the procedure from Morgan, et al 2011 (see references below).		To do a quick analysis of the data, we use ImageJ and the procedure from Morgan, et al 2011 (see references below).
		+
		+	Briefly, the procedure is as follows:
		+	# Open the GFP images in ImageJ as a series (File->Import->Image Sequence)
		+	# B

	==References==		==References==

Megan N McClean: /* References */

2012-06-06T15:38:35Z

References

←Older revision		Revision as of 15:38, 6 June 2012
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	* Miesenbock, G; De Angelis, DA; and JE Rothman (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins ''Nature''''' 394''' 192-195		* Miesenbock, G; De Angelis, DA; and JE Rothman (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins ''Nature''''' 394''' 192-195
	* Orij, R; Postmus, J; Beek, A; Brul, S; and G. Smits (2009) ''In vivo'' measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in ''Saccaromyces cerevisiae'' reveals a relation between intracellular pH and growth ''Microbiology'' '''155''' 268-278		* Orij, R; Postmus, J; Beek, A; Brul, S; and G. Smits (2009) ''In vivo'' measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in ''Saccaromyces cerevisiae'' reveals a relation between intracellular pH and growth ''Microbiology'' '''155''' 268-278
-	*Morgan, B; Sobotta, N; and T Dick (2011) Measuring EGSH and H<sub>2</sub>O<sub>2</sub> with roGFP2-based redox probes ''Free Radical Biology and Medicine''	+	*Morgan, B; Sobotta, N; and T Dick (2011) Measuring EGSH and H<sub>2</sub>O<sub>2</sub> with roGFP2-based redox probes ''Free Radical Biology and Medicine'' '''51''' 1943-1951

	==Notes==		==Notes==

Megan N McClean: /* References */

2012-06-06T15:36:24Z

References

←Older revision		Revision as of 15:36, 6 June 2012
Line 122:		Line 122:
	* Miesenbock, G; De Angelis, DA; and JE Rothman (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins ''Nature''''' 394''' 192-195		* Miesenbock, G; De Angelis, DA; and JE Rothman (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins ''Nature''''' 394''' 192-195
	* Orij, R; Postmus, J; Beek, A; Brul, S; and G. Smits (2009) ''In vivo'' measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in ''Saccaromyces cerevisiae'' reveals a relation between intracellular pH and growth ''Microbiology'' '''155''' 268-278		* Orij, R; Postmus, J; Beek, A; Brul, S; and G. Smits (2009) ''In vivo'' measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in ''Saccaromyces cerevisiae'' reveals a relation between intracellular pH and growth ''Microbiology'' '''155''' 268-278
-		+	*Morgan, B; Sobotta, N; and T Dick (2011) Measuring EGSH and H<sub>2</sub>O<sub>2</sub> with roGFP2-based redox probes ''Free Radical Biology and Medicine''

	==Notes==		==Notes==

Megan N McClean: /* Image Analysis */

2012-06-06T15:34:58Z

Image Analysis

←Older revision		Revision as of 15:34, 6 June 2012
Line 117:		Line 117:

	===Image Analysis===		===Image Analysis===
-	*Coming soon--Megan needs to write this part up	+	To do a quick analysis of the data, we use ImageJ and the procedure from Morgan, et al 2011 (see references below).
-		+

	==References==		==References==

Megan N McClean: /* Microscopy */

2012-06-06T15:34:19Z

Microscopy

←Older revision		Revision as of 15:34, 6 June 2012
Line 110:		Line 110:

	===Microscopy===		===Microscopy===
-	*Coming soon--Megan needs to ~~write~~ this ~~part up~~	+	The ratio of emission at 410nm excitation vs 470nm excitation is found by imaging with the pHluorin cube (Chroma D410/30x ET525/50m T495lpxr) and the GFP cube (ET470/40x ET525/50m T495LPXR). There are GFP and PHL imaging settings on the microscope that can be used with an ND acquisition. Make sure you put the cubes into the correct positions in the filter wheel (DAPI and Phluorin share the same spot, GFP and CY3 share the same position).
		+
		+	You will need to optimize exposure times and ND filter settings for your strain.
		+
		+	Please talk to Megan the first time you do this, she can show you what has been working for her.

	===Image Analysis===		===Image Analysis===

Megan N McClean: /* Notes */

2012-06-06T15:29:46Z

Notes

←Older revision		Revision as of 15:29, 6 June 2012
Line 123:		Line 123:
	==Notes==		==Notes==
	Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.		Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
-	*[[User:Megan N McClean\|Megan N McClean]] 10:33, 5 June 2012 (EDT)Currently, we assay pHluorin with two separate filter cubes (with different excitation filters, but the same emission filter). Eventually we will move the excitation filters and the emission filter onto the microscope's filter wheels, so that alignment of the filter cubes in the turret is not an issue and the to cut down on the switching time.	+	*[[User:Megan N McClean\|Megan N McClean]] 10:33, 5 June 2012 (EDT) Currently, we assay pHluorin with two separate filter cubes (with different excitation filters, but the same emission filter). Eventually we will move the excitation filters and the emission filter onto the microscope's filter wheels, so that alignment of the filter cubes in the turret is not an issue and the to cut down on the switching time.
-		+	*[[User:Megan N McClean\|Megan N McClean]] 11:29, 6 June 2012 (EDT) When I did an experiment on 6/5/2012, I noticed that after I permeabilized the cells and put them in appropriate pH buffers a large fraction of the cell population (this was with strain yMM1107 Mat alpha HAP1+ hoΔ::natMXAC-TDH3prom-Phluorin) went dark. Could this be because I over permeabilized the cells (incubated for 55minutes with digitonin instead of 30 minutes) or some other factor from the permeabilization and buffer protocol? In any event, this is something to think about optimizing for the future.
-		+

	==Contact==		==Contact==

Megan N McClean: /* Cell Preparation */

2012-06-05T15:08:09Z

Cell Preparation

←Older revision		Revision as of 15:08, 5 June 2012
Line 105:		Line 105:
	***Thaw a tube of [[McClean: ConA \| Concanavalin A]] from the -20°C freezer. Put 30μL in the bottom of each well, allow it to incubate for 5 minutes at room temperature, then aspirate away the solution.		***Thaw a tube of [[McClean: ConA \| Concanavalin A]] from the -20°C freezer. Put 30μL in the bottom of each well, allow it to incubate for 5 minutes at room temperature, then aspirate away the solution.
	**To each well of the 96-well plate at 190μL of the appropriate pH buffer from the above table.		**To each well of the 96-well plate at 190μL of the appropriate pH buffer from the above table.
-	**When the incubation is completed, spin down cells, resuspend in 5 mls of phosphate-buffered saline (PBS) and keep on ice. ~~Sonicate 250μL per microfuge~~ tube using program 1 on the Botstein sonicator.	+	**When the incubation is completed, spin down cells, resuspend in 5 mls of phosphate-buffered saline (PBS) and keep on ice.
		+	**For each well of the plate, spin down 500μL of cells in an Eppendorf tube. Then resuspend in 250μL of the appropriate pH buffer. Gently sonicate using program 1 on the Botstein sonicator.
	**To each well of the 96-well plate add 10μL of the sonicated cell culture. Allow cells to settle to the bottom of the well for 20-30 minutes at room temperature. Take to Nikon inverted scope for imaging (more detailed protocol to follow. We need to know a few days in advance when the plate will be ready to make sure we reserve microscope time).		**To each well of the 96-well plate add 10μL of the sonicated cell culture. Allow cells to settle to the bottom of the well for 20-30 minutes at room temperature. Take to Nikon inverted scope for imaging (more detailed protocol to follow. We need to know a few days in advance when the plate will be ready to make sure we reserve microscope time).