Live-cell Microscopy and Fluorescence-based Measurement of Luminal pH in Intracellular Organelles
Luminal pH is an important functional feature of intracellular organelles. Acidification of the lumen of organelles such as endosomes, lysosomes, and the Golgi apparatus plays a critical role in fundamental cellular processes. As such, measurement of the luminal pH of these organelles has relevance...
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Frontiers Media S.A.
2017-08-01
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| Series: | Frontiers in Cell and Developmental Biology |
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| Online Access: | http://journal.frontiersin.org/article/10.3389/fcell.2017.00071/full |
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| author | Li Ma Li Ma Qing Ouyang Qing Ouyang Gordon C. Werthmann Heather M. Thompson Heather M. Thompson Heather M. Thompson Eric M. Morrow Eric M. Morrow Eric M. Morrow |
| author_facet | Li Ma Li Ma Qing Ouyang Qing Ouyang Gordon C. Werthmann Heather M. Thompson Heather M. Thompson Heather M. Thompson Eric M. Morrow Eric M. Morrow Eric M. Morrow |
| author_sort | Li Ma |
| collection | DOAJ |
| description | Luminal pH is an important functional feature of intracellular organelles. Acidification of the lumen of organelles such as endosomes, lysosomes, and the Golgi apparatus plays a critical role in fundamental cellular processes. As such, measurement of the luminal pH of these organelles has relevance to both basic research and translational research. At the same time, accurate measurement of intraorganellar pH in living cells can be challenging and may be a limiting hurdle for research in some areas. Here, we describe three powerful methods to measure rigorously the luminal pH of different intracellular organelles, focusing on endosomes, lysosomes, and the Golgi apparatus. The described methods are based on live imaging of pH-sensitive fluorescent probes and include: (1) A protocol based on quantitative, ratiometric measurement of endocytosis of pH-sensitive and pH-insensitive fluorescent conjugates of transferrin; (2) A protocol for the use of proteins tagged with a ratiometric variant of the pH-sensitive intrinsically fluorescent protein pHluorin; and (3) A protocol using the fluorescent dye LysoSensor™. We describe necessary reagents, key procedures, and methods and equipment for data acquisition and analysis. Examples of implementation of the protocols are provided for cultured cells derived from a cancer cell line and for primary cultures of mouse hippocampal neurons. In addition, we present strengths and weaknesses of the different described intraorganellar pH measurement methods. These protocols are likely to be of benefit to many researchers, from basic scientists to those conducting translational research with a focus on diseases in patient-derived cells. |
| first_indexed | 2024-12-10T19:09:01Z |
| format | Article |
| id | doaj.art-7bd40a01910a45a5befc558ee6959b5a |
| institution | Directory Open Access Journal |
| issn | 2296-634X |
| language | English |
| last_indexed | 2024-12-10T19:09:01Z |
| publishDate | 2017-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cell and Developmental Biology |
| spelling | doaj.art-7bd40a01910a45a5befc558ee6959b5a2022-12-22T01:36:49ZengFrontiers Media S.A.Frontiers in Cell and Developmental Biology2296-634X2017-08-01510.3389/fcell.2017.00071259250Live-cell Microscopy and Fluorescence-based Measurement of Luminal pH in Intracellular OrganellesLi Ma0Li Ma1Qing Ouyang2Qing Ouyang3Gordon C. Werthmann4Heather M. Thompson5Heather M. Thompson6Heather M. Thompson7Eric M. Morrow8Eric M. Morrow9Eric M. Morrow10Department of Molecular Biology, Cell Biology and Biochemistry, Brown UniversityProvidence, RI, United StatesBrown Institute for Brain Science, Brown UniversityProvidence, RI, United StatesDepartment of Molecular Biology, Cell Biology and Biochemistry, Brown UniversityProvidence, RI, United StatesBrown Institute for Brain Science, Brown UniversityProvidence, RI, United StatesDepartment of Molecular Biology, Cell Biology and Biochemistry, Brown UniversityProvidence, RI, United StatesDepartment of Molecular Biology, Cell Biology and Biochemistry, Brown UniversityProvidence, RI, United StatesBrown Institute for Brain Science, Brown UniversityProvidence, RI, United StatesHassenfeld Child Health Innovation Institute, Brown UniversityProvidence, RI, United StatesDepartment of Molecular Biology, Cell Biology and Biochemistry, Brown UniversityProvidence, RI, United StatesBrown Institute for Brain Science, Brown UniversityProvidence, RI, United StatesHassenfeld Child Health Innovation Institute, Brown UniversityProvidence, RI, United StatesLuminal pH is an important functional feature of intracellular organelles. Acidification of the lumen of organelles such as endosomes, lysosomes, and the Golgi apparatus plays a critical role in fundamental cellular processes. As such, measurement of the luminal pH of these organelles has relevance to both basic research and translational research. At the same time, accurate measurement of intraorganellar pH in living cells can be challenging and may be a limiting hurdle for research in some areas. Here, we describe three powerful methods to measure rigorously the luminal pH of different intracellular organelles, focusing on endosomes, lysosomes, and the Golgi apparatus. The described methods are based on live imaging of pH-sensitive fluorescent probes and include: (1) A protocol based on quantitative, ratiometric measurement of endocytosis of pH-sensitive and pH-insensitive fluorescent conjugates of transferrin; (2) A protocol for the use of proteins tagged with a ratiometric variant of the pH-sensitive intrinsically fluorescent protein pHluorin; and (3) A protocol using the fluorescent dye LysoSensor™. We describe necessary reagents, key procedures, and methods and equipment for data acquisition and analysis. Examples of implementation of the protocols are provided for cultured cells derived from a cancer cell line and for primary cultures of mouse hippocampal neurons. In addition, we present strengths and weaknesses of the different described intraorganellar pH measurement methods. These protocols are likely to be of benefit to many researchers, from basic scientists to those conducting translational research with a focus on diseases in patient-derived cells.http://journal.frontiersin.org/article/10.3389/fcell.2017.00071/fullendosomefluorescenceGolgilysosomeorganellepHluorin |
| spellingShingle | Li Ma Li Ma Qing Ouyang Qing Ouyang Gordon C. Werthmann Heather M. Thompson Heather M. Thompson Heather M. Thompson Eric M. Morrow Eric M. Morrow Eric M. Morrow Live-cell Microscopy and Fluorescence-based Measurement of Luminal pH in Intracellular Organelles Frontiers in Cell and Developmental Biology endosome fluorescence Golgi lysosome organelle pHluorin |
| title | Live-cell Microscopy and Fluorescence-based Measurement of Luminal pH in Intracellular Organelles |
| title_full | Live-cell Microscopy and Fluorescence-based Measurement of Luminal pH in Intracellular Organelles |
| title_fullStr | Live-cell Microscopy and Fluorescence-based Measurement of Luminal pH in Intracellular Organelles |
| title_full_unstemmed | Live-cell Microscopy and Fluorescence-based Measurement of Luminal pH in Intracellular Organelles |
| title_short | Live-cell Microscopy and Fluorescence-based Measurement of Luminal pH in Intracellular Organelles |
| title_sort | live cell microscopy and fluorescence based measurement of luminal ph in intracellular organelles |
| topic | endosome fluorescence Golgi lysosome organelle pHluorin |
| url | http://journal.frontiersin.org/article/10.3389/fcell.2017.00071/full |
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