Background

The intracellular pH of yeast is correlated with characteristics like viability and growth rate, and the regulation of intracellular pH consumes a large proportion of cellular energetic resources (Orij et al., 2011). However, intracellular pH can change rapidly and is highly environmentally sensitive, so it is crucial to have a fast, minimally perturbative method of measurement for this important aspect of cell physiology.

Genetically-encoded biosensors that convert local concentrations of a compound of interest into fluorescent readouts have revolutionized our ability to characterize the intracellular environment. For some sensors, the absolute fluorescence intensity is correlated with the readout. This can be a problem when performing in-cell measurements, since the fluorescence depends both on the sensor expression level, which varies cell to cell, and the characteristic of interest. Ratiometric sensors, which depend on the ratio of fluorescence in two different parts of the spectrum in the same fluorophore, do not suffer from this drawback. One such sensor, pHluorin (Miesenböck et al., 1998), is a pH-sensitive fluorescent biosensor based on GFP that can be used to measure intracellular pH; the emission intensity (measured around 520 nm) after excitation in the near-UV and blue (405 and 488 nm typically) varies with pH such that the ratio of emission intensities can be related to pH.

In this protocol, we outline how to use pHluorin to measure intracellular pH in living budding yeast cells using flow cytometry. Flow cytometry is a method that combines microfluidic focusing and optical interrogation to measure the fluorescence of single cells in liquid culture with little to no special sample preparation required. Others have also used this method to measure intracellular pH in yeast cells (Weigert et al., 2009; Valkonen et al., 2013). The advantage of flow cytometry is that it is much higher throughput than microscopy-based methods (Bagar et al., 2009; Orij et al., 2009), while still being able to characterize individual cells and measure the fluorescence of multiple fluorophores. This access to both single-cell measurements and enough data to generate population-level statistics with a great deal of confidence is highly valuable. One potential downside to using flow cytometry to analyze the fluorescence of biosensors is that the spatial distribution of the fluorophore within the cell is not accessible (at least with traditional flow cytometry), as only a single, average fluorescence value in each channel is reported for each event (cell). However, for intracellular pH measurements in particular, the variation in any compartment (here, the cytosol, although pHluorin can be targeted to other organelles) (Orij et al., 2009) is expected to be minimal due to the unique properties of proton exchange in buffered aqueous solutions such as the cellular interior (Boron, 2004).

The outline of a typical experiment is illustrated in Figure 1. Cells expressing pHluorin are suspended in buffer of known pH and an ionophore, in this case nigericin, is added. This addition makes the cell membrane permeable to protons and thus equilibrates the intracellular and extracellular pH. The ratiometric fluorescence of pHluorin is measured for these cells with known intracellular pH, and then these data are used to construct a calibration curve that can be used to convert measured fluorescence ratios in other cells to real intracellular pH values.


Figure 1. Flowchart of Protocol. Experimental steps are colored blue, analysis steps are in green. Optional steps are in gray text.