McClean:Phluorin Calibration

Overview

Ratiometric pHluorin is a pH-sensitive GFP derivative. See Miesenbock, et al 1998 for the original description of this protein. Wild-type green fluorescent protein (GFP) exists in two conformations and therefore has a bimodal excitation spectrum with peaks at 395nm and 475nm. The authors took advantage of these conformation states, and with some clever amino-acid substitutions facilitated pH-dependent switching between the states. Thus, the relative emission of pHluorin when excited at 395nm vs 475nm is dependent on pH.

This protocol describes how to measure a calibration curve for pHluorin in your yeast strain of interest. The relative emission at 395nm or 475nm excitation is measured for permeabilized cells in buffers over a range of pH. This protocol was adapted from Orij 2009. See their paper (Figure 2a) for a good example of what your calibration curve should look like.

Materials

• Strain of interest
• Low Fluorescence Media with appropriate amino acids for your strain
• Concanavalin A Solution for Microfluidics
• Digitonin (Acros Organics, #407565000)
• Phosphate Buffered Saline, pH 7
• Buffers (see below)
• 96-well glass-bottom plates (Nunc, #265300) for imaging

Buffers

We use a citric acid/Na₂HPO₄ buffer to create pH values ranging from approximately pH 5 to pH 9. Tina Hansen made the following convenient chart when she tested this protocol. You should probably test the pH of your buffers (as Tina did) to make sure that you know what the actual pH is (instead of relying on what it "should" be based on a buffer table).

pH actual	Label on tube (pH expected)	x ml 0.1M-citric acid (for 100ml buffer)	y ml 0.2-Na2HPO4 (for 100ml buffer)	x ml 0.1M-citric acid (for 10ml buffer)	y ml 0.2-Na2HPO4 (for 10ml buffer)
4.79	3.2	57	43	5.7	4.3
4.97	3.4	56	44	5.6	4.4
4.98	3.6	55	45	5.5	4.5
5.06	3.8	54	46	5.4	4.6
5.19	4	53	47	5.3	4.7
5.24	4.2	52	48	5.2	4.8
5.45	4.4	51	49	5.1	4.9
5.49	4.6	50	50	5	5
5.55	4.8	49.3	50.7	4.93	5.07
5.6	5	48.5	51.5	4.85	5.15
5.8	5.2	46.4	53.6	4.64	5.36
6	5.4	44.25	55.75	4.425	5.575
6.1	5.6	42	58	4.2	5.8
6.3	5.8	39.55	60.45	3.955	6.045
6.57	6	36.85	63.15	3.685	6.315
6.68	6.2	33.9	66.1	3.39	6.61
6.78	6.4	30.75	69.25	3.075	6.925
6.98	6.6	27.25	72.75	2.725	7.275
7.13	6.8	22.75	77.25	2.275	7.725
7.43	7	17.65	82.35	1.765	8.235
7.53	7.2	13.05	86.95	1.305	8.695
7.7	7.4	9.15	90.85	0.915	9.085
7.9	7.6	6.35	93.65	0.635	9.365
7.93	7.8	5.65	94.35	0.565	9.435
8.04	8	4.95	95.05	0.495	9.505
8.03	8.2	4.25	95.75	0.425	9.575
8.26	8.4	3.55	96.45	0.355	9.645
8.26	8.6	2.85	97.15	0.285	9.715
8.33	8.8	2.15	97.85	0.215	9.785
8.36	9	1.45	98.55	0.145	9.855
8.63	9.2	0.75	99.25	0.075	9.925
8.7	9.4	0.05	99.95	0.005	9.995
8.76	stock bottle	0	100	0	10

Protocol

Cell Preparation

• Day 1:
  • Grow your strain of interest in 5mls of Low Fluorescence Media (LFM) overnight to saturation
• Day 2:
  • In the morning, reinoculate 250μL of your saturated overnight culture into 25mls of fresh media in a Erlenmeyer flask. Grow with shaking at 30°C until cells reach an OD660 of ~1.
  • Once cells reach an appropriate OD, spin down 10 mls of the cell culture and resuspend in 5mls of PBS containing 100μg/ml Digitonin (0.1% w/v) in a culture tube. Incubate on the wheel (with rotation) at 30°C for 30 minutes.
  • While cells are incubating, prepare a 96-well glass-bottom plate for imaging. Prepare one well per pH value that you would like to assay with your cells. To prepare the wells:
    • Thaw a tube of 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.
  • 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).

Microscopy

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

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:

1. Open the GFP images in ImageJ as a series (File->Import->Image Sequence)
2. Background subtract the images (Process->Subtract Background; Usually for yeast keeping the rolling ball radius at 50 works well)
3. Convert the image to 32-bit (Image->Type->32-bit)
4. Repeat steps 1-3 with the Phluorin cube images
5. 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)
6. 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)
7. 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

• 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
• Morgan, B; Sobotta, N; and T Dick (2011) Measuring EGSH and H₂O₂ with roGFP2-based redox probes Free Radical Biology and Medicine 51 1943-1951

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 '''*~~~~''': to the beginning of your tip.

• 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.
• 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.
• 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

• Megan N McClean 14:01, 05 June 12(EDT)

or instead, discuss this protocol.