The overall goal of the following experiment is to simultaneously measure vesicular and cytosolic pH in living cells by confocal microscopy. This is achieved by first incubating the cells with the pH sensing probes, HPTS and SNF F1, which label the endosomal and lysosomal compartments and the cytosol respectively. Next, the cells are placed in an environmentally controlled chamber under a confocal microscope, and the software is set to measure the fluorescence of the probes in the chamber.
The cells are incubated with different pH calibrated solutions. A pH standard curve is constructed using a non-linear regression. Exponential equations are determined for both probes and are subsequently used to convert fluorescence intensities of samples into pH values.
This method allows the determination of vesicular and cytosolic pH from samples after various treatments and can enable the study of the regulation of intracellular pH during biological processes. The main advantage of this technique over existing methods for intracellular pH measurement is that our method enables the measurement of intracellular pH dynamics in living cells while eliminating artifacts and background noise During imaging, demonstrating the procedure will be fess lucier, the PhD student who has developed this method For cellular pH calibration. It is necessary to create a series of five 50 milliliter solutions from pH 5.5 to 7.5 in 0.5 increments.
Add one normal potassium hydroxide or hydrochloric acid dropwise to set the pH values. Then to each pH calibrated solution add 0.05 milliliters of the IOR gersin. At 10 millimolar for a final concentration of 10 micromolar risin will increase cell membrane permeability in the presence of depolarizing concentrations of extracellular potassium under a hood seed cells in 35 millimeter culture dishes with two milliliters of medium containing 10%fetal bovine serum and antibiotics with the goal of achieving 50%confluence in 24 hours of incubation at 37 degrees Celsius.
After seeding all the required plates for the experiment seed an additional five plates for the calibration. Incubate these plates for 24 hours, 24 hours later, and 16 hours before imaging. Add two microliters of the organelle label HPTS at one molar for a final concentration of one millimolar HPTS in solution on the next morning, about three hours.
Before imaging. Remove the HPTS by performing two washes with PBS. Then add two milliliters of serum free medium and allow the cells to incubate for at least two more hours.
Just before imaging incubate the cells with five micromolar snf, one by adding 10 microliters of one millimolar snf, one following the SNF one incubation period. Wash the cells twice with PBS and add two milliliters of serum free medium, then proceed with imaging. The cells.
Imaging of the cells should be accomplished with a spectral inverted scanning confocal microscope, equipped with a motorized stage and environmental chamber. When using plastic petri dishes, a 40 x objective is the most appropriate. The software should be adjusted for two virtual channel sets in the software, adjust the excitation wavelength and emission wavelengths.
For HPTS and SNF one, the confocal aperture is set to 175 microns by default, and optimal aperture needs to be manually set at 300 microns. Once the chamber has warmed to 37 degrees Celsius, start the calibration, wash a plate of cells twice more using PBS, and then replace the medium with the first calibration solution. Now set the cells into the chamber and calibrate both pH sensitive dyes acquire up to 30 time lapse images at one minute intervals.
Each image stack can be collected at 0.4 microns thick and 800 square pixels. And between images, the shutter must be programmed to remain closed. Note the fluorescent intensity every minute, which will typically stabilize around 20 minutes.
Once fluorescent stabilizes, take images from four or five fields containing 15 to 20 cells, and both probes. These images are used for the curve calculations. Repeat this process for each calibration solution.
Then proceed with imaging the experimental condition plates for which no washes are needed In analyzing each image, first, digitally subtract out the background extracellular fluorescence. Then select labeled vesicles and cytosol from stored images using a binary mask. For each pH sensing probe, fluorescence intensities are measured on a scale from zero to 4, 095.
Calculate the mean intensity for all pixels in the organelle or cytoplasm. Using these values, calculate fluorescence ratios for HPTS and for snf. One for calibration curves plot the fluorescence ratios obtained for each calibration solution as a function of pH calibration curve fitting should be done using an exponential equation.
Lastly, use the calibration curves to transform the ratios into pH values and thus obtain cytosolic and organelle pH values in living cells under experimental conditions. Clear evidence was obtained that HPTS labels both endosomal and lysosomal compartments by cos staining with Alexa 5 46 conjugated transferrin to stain endosomes or with a fluorescent ly atrophic probe. Lyo tracker.
Deep red time-lapse images were collected for each probe as described in HT 10 80 cells. Five calibration solutions were tested fluorescence approach stability after 15 minutes in the solutions such as with pH six and pH 7.5 solutions in the calibration curve for a given pH, the data represents the ratio of fluorescence intensities at 558 to 405 nanometers for HPTS or 644 to 584 nanometers for SNF one. Both curves were fitted with an exponential equation.
The efficiency of the method in live HT 10 80 was evaluated using ammonium chloride, a weak base that increases intracellular pH. The base increased the pH of both the cytosol and the vesicles. The method was also evaluated using B mycin, A one, an inhibitor of va a TPAs bamy, A one induced alkalinization of both endosomal and lysosomal compartments, but did not alter cytosolic pH.
Lastly, EIPA, an inhibitor of NHE one was tested. It acidified the cytosol down to a pH of 6.62, but did not affect pH. So far.
The study of cellular pH homeostasis has been limited both by rapid pH changes occurring within cells, as well as a lack of tools to studies pH within cells. We describe today a simple step-by-step approach to simultaneously measure both cytosolic and endosomal pH in single living cells. This is especially important for the study of cellular pH homeostasis in cells where mutations or drugs can affect the influx and influx of protons within the endosomal compartment.
