The overall goal of the following experiment is to measure vacuolar and cytosolic pH in living yeast cells and to observe how pH changes with alterations in extracellular conditions. This is achieved by introducing pH sensitive fluorescent probes to the VA and cytosol of yeast cells. The dye B-C-E-C-F-A-M is introduced to the cells in order to measure ular pH and a pH sensitive GFP Florin is expressed from a plasmid in the cells in order to measure cytosolic pH.
As a second step, fluorescently labeled cells are washed and resuspended at high density in growth. Medium lacking glucose. Aliquots of the suspension are distributed to calibration buffers of different pH to create the calibration curve.
Next, another portion of the suspension is diluted in a weak buffer and the fluorescence of the suspension is continuously monitored in a fluorimeter before and after addition of glucose or other additives. In order to determine valar or cytosolic pH responses in vivo results are obtained that show time and condition dependent pH conditions in the experimental sample based on ratio metric fluorescence ratios calibrated from the calibration samples. I'm Dr.Patty Kane, professor of biochemistry and molecular biology at SUNY Upstate Medical University.
This method can help answer key questions in pa, the field of pH regulations, such as how cells respond to environmental changes and how much different pumps and transporters contribute to cytosolic and ular pH control. To begin this procedure, grow a 50 milliliter liquid culture of wild type yeast cells in the desired medium. In this case, YEPD medium buffered to pH five with 50 millimolar MES overnight.
Once an absorbance of 0.8 has been achieved, pellet the yeast cells by centrifugation at 3000 times G for five minutes. Then remove the media and resus resuspend the pellet in 0.6 milliliters of fresh growth medium. Next, transfer the suspension to a micro centrifuge tube that has been weighed previously and palate again at 2000 times G for 60 seconds.
Remove the supernatant as completely as possible and then weigh the cell pellet. Resuspend the pellet to a final density of 0.5 grams per milliliter in culture medium. Next, add B-C-E-C-F-M to the cell suspension at a final concentration of 50 micromolar from a 12 millimolar stock prepared in DMSO mix well and then incubate the cells at 30 degrees Celsius for 30 minutes on a spinning drum rotor.
During this time, the cells will uptake the B-C-E-C-F-A am as shown here while the cells are incubating. Prepare calibration buffers for measurement of ular pH in wild type cells. Prepare calibration mixtures of pH 5.5, 5.76, 6.3, and 6.5.
Next, aliquot two milliliters of calibration buffer for each pH into 15 milliliter conical tubes. Then add menin and Niger to give final concentrations of 110 micromolar and 15 micromolar respectively and vortex. When the incubation time is completed, pellet the cell suspension by centrifugation for 30 seconds at 2000 times G Resus, suspend the cells in one milliliter of growth, medium, lacking glucose, and centrifuge again for 30 seconds.
At 2000 times G, remove the media from the pellet and rinse once more. Then resuspend the pellet in 200 microliters of growth, medium without glucose, and place it on ice. Next, add 20 microliters of the cell suspension to each pH calibration tube.
Incubate all tubes at 30 degrees Celsius for 30 to 60 minutes using a roller drum to allow them to equilibrate. Store the remaining cells on ice for use within 30 minutes while the cells are incubating. Set the fluorimeter to alternately measure at excitation wavelengths 450 nanometers and 490 nanometers, both with an emission wavelength of 535 nanometers.
Also, prewarm the sample chamber temperature to 30 degrees Celsius. Begin taking measurements starting with the experimental sample. The experimental sample consists of 1.96 milliliters of one millimolar MES at pH 5.0.
Combined with 20 microliters of cell suspension. Perform all measurements with continuous stirring of the mixture in the qve. Collect continuous kinetic data by taking measurements every six seconds for five minutes.
Then add glucose to a final glucose concentration of 50 millimolar, and continue measurements for an additional five to 10 minutes After the experimental measurements are complete, remove the calibration tubes from the 30 degrees Celsius roller drum and transfer the entire two millimeter volume for each to the fluorimeter vete. Measure fluorescence at the same settings every five seconds over a total of 30 seconds for each sample. Next, export the fluorescence data to Microsoft Excel.
Obtain a calibration curve by calculating the ratio of fluorescence at 490 nanometers to 450 nanometers for each calibration mixture. The fluorescence ratio is then plotted versus pH to get a calibration curve. To begin cytosolic pH monitoring, transform the desired yeast strain with the yeast florin plasmid by standard protocols to produce cells that fluoresce in the cytosol like the ones shown here.
Plate the transformed cells on an agar plate lacking uracil to select for transformed cells. Then pick transformants from the plate and inoculate starter cultures. Also lacking uracil.
Expand the transfected cells in a 50 milliliter liquid culture of growth medium, lacking uracil to mid log phase with an OD 600 of close to 0.8. Next, prepare calibration standards of pH six, 6.57, 7.5, and eight as shown in the previous section. This range is appropriate for cytosolic pH measurements.
Aliquot two milliliters of each calibration buffer into a fresh 15 milliliter conical tube, menin and Niger to give final concentrations of 110 micromolar and 15 micromolar respectively, and mix well. Once the standards have been prepared, harvest the cells by centrifugation and resuspend the pellet in 600 microliters of the growth medium. Transfer the cells to a weighed micro centrifuge tube and pellet them by centrifugation at 5, 000 RPM for 30 seconds.
Then resuspend the pellet in one milliliter of growth, medium without glucose or uracil pellet cells again and repeat to rinse the cells. One more time. After the final centrifugation, remove the supernatant as thoroughly as possible and weigh the cell palate.
This time, resus suspend cells to a final density of 0.5 grams per milliliter using growth medium without uracil or glucose. Add 20 microliters of cell suspension to each tube of calibration buffer and mix well on a vortex mixer. Incubate the vials at 30 degrees Celsius on a spinning drum rotor for 60 minutes During incubation, set up the fluorimeter at excitation wavelengths of 405 and 485 nanometers and an emission wavelength of 508 nanometers.
Prepare the experimental sample by adding 1.96 milliliters of one millimolar MES at pH 5.0 combined with 20 microliters of cell suspension, and collect kinetic data by reading the sample every six seconds for five minutes. Then add glucose and continue measurements for an additional five to 10 minutes After the experimental measurements are complete, measure the calibration sample fluorescence at the same settings every five seconds over a total of 30 seconds for each sample. Then export the fluorescence data as before to Microsoft Excel.
Obtain a calibration curve by calculating the ratio of fluorescence at 405 nanometers to 485 nanometers and plot it versus pH to get a calibration curve. Finally, convert the experimental data to fluorescence ratio and calculate pH using the standard curve. Cytosolic pH versus fluorescence ratio is linear over the range of most cytosolic pH measurements making this conversion easy.
Calibration curves like the one shown here are typical for wild type yeast cells using the protocol shown here. Although the relationship between the fluorescence ratio and vascular pH is nonlinear over a wide pH range, it is nearly linear in the relevant range for these experimental measurements. ULAR pH changes resulting from the addition of glucose to glucose deprived cells are shown here.
After glucose re edition at five minutes, the pH decreases presumably as a result of valar APAs activation through reassembly. The addition of 50 millimolar potassium chloride three minutes after adding glucose increases the pH slightly. Unlike the valar pH calibration curve, the cytosolic fluorescence ratio is linear to the cytosolic pH over a wide range.
The kinetics of cytosolic pH change in wild type yeast cells due to the addition of glucose, decreases at first, but then increases to a pH between 7.2 and 7.4. The initial pH decrease reflects the initiation of glucose metabolism, and the subsequent pH increase corresponds to activation of proton export mechanisms that remove cytosolic protons. While attempting this procedure, it's important to remember that intracellular pH values and responses are sensitive to many extracellular factors such as growth stage of the cells and pH of the growth medium.
These potential variables should be controlled as much as possible to achieve reproducible results. Thank you for your time and interest.
