McClean:Phluorin Calibration

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(Buffers)
Line 12: Line 12:
* Phosphate Buffered Saline, pH 7
* Phosphate Buffered Saline, pH 7
* Buffers (see below)
* Buffers (see below)
 +
* 96-well glass-bottom plates (Nunc, #265300) for imaging
==Buffers==
==Buffers==
Line 93: Line 94:
==Protocol==
==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 conA () 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 conA.
 +
**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.
 +
**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===
 +
*Coming soon--Megan needs to write this part up
 +
 +
===Image Analysis===
 +
*Coming soon--Megan needs to write this part up

Revision as of 11:04, 5 June 2012


Contents

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
  • 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)
4.793.257435.74.3
4.973.456445.64.4
4.983.655455.54.5
5.063.854465.44.6
5.19453475.34.7
5.244.252485.24.8
5.454.451495.14.9
5.494.6505055
5.554.849.350.74.935.07
5.6548.551.54.855.15
5.85.246.453.64.645.36
65.444.2555.754.4255.575
6.15.642584.25.8
6.35.839.5560.453.9556.045
6.57636.8563.153.6856.315
6.686.233.966.13.396.61
6.786.430.7569.253.0756.925
6.986.627.2572.752.7257.275
7.136.822.7577.252.2757.725
7.43717.6582.351.7658.235
7.537.213.0586.951.3058.695
7.77.49.1590.850.9159.085
7.97.66.3593.650.6359.365
7.937.85.6594.350.5659.435
8.0484.9595.050.4959.505
8.038.24.2595.750.4259.575
8.268.43.5596.450.3559.645
8.268.62.8597.150.2859.715
8.338.82.1597.850.2159.785
8.3691.4598.550.1459.855
8.639.20.7599.250.0759.925
8.79.40.0599.950.0059.995
8.76stock bottle0100010

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 conA () 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 conA.
    • 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.
    • 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

  • Coming soon--Megan needs to write this part up

Image Analysis

  • Coming soon--Megan needs to write this part up


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


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.


Contact

or instead, discuss this protocol.

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