Editing Intracellular pH (section)

== Measurement ==
There are several common ways in which intracellular pH (pH<sub>i</sub>) can be measured including with a microelectrode, dye that is sensitive to pH, or with nuclear magnetic resonance techniques.<ref name=":3">{{cite journal | vauthors = Roos A, Boron WF | title = Intracellular pH | journal = Physiological Reviews | volume = 61 | issue = 2 | pages = 296–434 | date = April 1981 | pmid = 7012859 | doi = 10.1152/physrev.1981.61.2.296 }}</ref><ref name=":1" /> For measuring pH inside of organelles, a technique utilizing pH-sensitive green fluorescent proteins (GFPs) may be used.<ref name=":5">{{cite journal | vauthors = Roberts TM, Rudolf F, Meyer A, Pellaux R, Whitehead E, Panke S, Held M | title = Corrigendum: Identification and Characterisation of a pH-stable GFP | journal = Scientific Reports | volume = 8 | pages = 46976 | date = May 2018 | pmid = 29769631 | pmc = 5956236 | doi = 10.1038/srep46976 | bibcode = 2018NatSR...846976R }}</ref>

Overall, all three methods have their own advantages and disadvantages. Using dyes is perhaps the easiest and fairly precise, while NMR presents the challenge of being relatively less precise.<ref name=":3" /> Furthermore, using a microelectrode may be challenging in situations where the cells are too small, or the intactness of the cell membrane should remain undisturbed.<ref name=":1" /> GFPs are unique in that they provide a noninvasive way of determining pH inside different organelles, yet this method is not the most quantitatively precise way of determining pH.<ref name=":6" />

=== Microelectrode ===
The microelectrode method for measuring pH<sub>i</sub> consists of placing a very small electrode into the cell’s cytosol by making a very small hole in the plasma membrane of the cell.<ref name=":1" /> Since the microelectrode has fluid with a high H+ concentration inside, relative to the outside of the electrode, there is a potential created due to the pH discrepancy between the inside and outside of the electrode.<ref name=":3" /><ref name=":1" /> From this voltage difference, and a predetermined pH for the fluid inside the electrode, one can determine the intracellular pH (pH<sub>i</sub>) of the cell of interest.<ref name=":1">{{cite book | vauthors = Loiselle FB, Casey JR | title = Membrane Transporters in Drug Discovery and Development | chapter = Measurement of Intracellular pH | volume = 637 | pages = 311–31 | date = 2010 | pmid = 20419443 | doi = 10.1007/978-1-60761-700-6_17 | isbn = 978-1-60761-699-3 | series = Methods in Molecular Biology }}
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=== Fluorescence spectroscopy ===
Another way to measure Intracellular pH (pH<sub>i</sub>) is with dyes that are sensitive to pH, and fluoresce differently at various pH values.<ref name=":4" /><ref>{{cite journal | vauthors = Specht EA, Braselmann E, Palmer AE | title = A Critical and Comparative Review of Fluorescent Tools for Live-Cell Imaging | journal = Annual Review of Physiology | volume = 79 | pages = 93–117 | date = February 2017 | pmid = 27860833 | doi = 10.1146/annurev-physiol-022516-034055 | pmc = 12034319 }}</ref> This technique, which makes use of fluorescence spectroscopy, consists of adding this special dye to the cytosol of a cell.<ref name=":3" /><ref name=":1" /> By exciting the dye in the cell with energy from light, and measuring the wavelength of light released by the photon as it returns to its native energy state, one can determine the type of dye present, and relate that to the intracellular pH of the given cell.<ref name=":3" /><ref name=":1" />

=== Nuclear magnetic resonance ===
In addition to using pH-sensitive electrodes and dyes to measure pH<sub>i</sub>, Nuclear Magnetic Resonance (NMR) spectroscopy can also be used to quantify pH<sub>i</sub>.<ref name=":1" /> NMR, typically speaking, reveals information about the inside of a cell by placing the cell in an environment with a potent magnetic field.<ref name=":3" /><ref name=":1" /> Based on the ratio between the concentrations of protonated, compared to deprotonated, forms of phosphate compounds in a given cell, the internal pH of the cell can be determined.<ref name=":3" /> Additionally, NMR may also be used to reveal the presence of intracellular sodium, which can also provide information about the pH<sub>i</sub>.<ref>{{cite journal | vauthors = Eliav U, Navon G | title = Sodium NMR/MRI for anisotropic systems | journal = NMR in Biomedicine | volume = 29 | issue = 2 | pages = 144–52 | date = February 2016 | pmid = 26105084 | doi = 10.1002/nbm.3331 | s2cid = 29964258 }}</ref>

Using NMR Spectroscopy, it has been determined that [[lymphocyte]]s maintain a constant internal pH of 7.17± 0.06, though, like all cells, the intracellular pH changes in the same direction as extracellular pH.<ref>{{cite journal | vauthors = Deutsch C, Taylor JS, Wilson DF | title = Regulation of intracellular pH by human peripheral blood lymphocytes as measured by 19F NMR | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 79 | issue = 24 | pages = 7944–8 | date = December 1982 | pmid = 6961462 | pmc = 347466 | doi = 10.1073/pnas.79.24.7944 | bibcode = 1982PNAS...79.7944D | doi-access = free }}</ref>

=== pH-sensitive GFPs ===
To determine the pH inside organelles, pH-sensitive GFPs are often used as part of a noninvasive and effective technique.<ref name=":5" /> By using cDNA as a template along with the appropriate primers, the GFP gene can be expressed in the cytosol, and the proteins produced can target specific regions within the cell, such as the mitochondria, golgi apparatus, cytoplasm, and endoplasmic reticulum.<ref name=":6">{{cite journal | vauthors = Kneen M, Farinas J, Li Y, Verkman AS | title = Green fluorescent protein as a noninvasive intracellular pH indicator | journal = Biophysical Journal | volume = 74 | issue = 3 | pages = 1591–9 | date = March 1998 | pmid = 9512054 | pmc = 1299504 | doi = 10.1016/S0006-3495(98)77870-1 | bibcode = 1998BpJ....74.1591K }}</ref> If certain GFP mutants that are highly sensitive to pH in intracellular environments are used in these experiments, the relative amount of resulting fluorescence can reveal the approximate surrounding pH.<ref name=":6" /><ref>{{cite journal | vauthors = Rizzuto R, Brini M, Pizzo P, Murgia M, Pozzan T | title = Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells | journal = Current Biology | volume = 5 | issue = 6 | pages = 635–42 | date = June 1995 | pmid = 7552174 | doi = 10.1016/s0960-9822(95)00128-x | s2cid = 13970185 | doi-access = free | bibcode = 1995CBio....5..635R }}</ref>