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Analysis of Lysosomal pH by Flow Cytometry Using FITC-Dextran Loaded Cells
Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Pathology and Clinical Genetics.ORCID iD: 0000-0003-4075-159X
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
2017 (English)In: Lysosomes: Methods and Protocols / [ed] Karin Öllinger;Hanna Appelqvist, Humana Press, 2017, Vol. 1594, p. 179-189Chapter in book (Refereed)
Abstract [en]

The acidic environment of the lysosomal lumen provides an optimal milieu for the acid hydrolases and is also essential for fusion/fission of endo-lysosomal compartments and sorting of cargo. Evidence suggests that maintaining lysosomal acidity is essential to avoid disease. In this chapter, we describe a protocol for analyzing the lysosomal pH in cultured cells using the fluorescent probe fluorescein isothiocyanate (FITC)-dextran together with a dual-emission ratiometric technique suitable for flow cytometry. Fluorescence-labeled dextran is endocytosed and accumulated in the lysosomal compartment. FITC shows a pH-dependent variation in fluorescence when analyzed at maximum emission wavelength and no variation when analyzing at the isosbestic point, thereby the ratio can be used to determine the lysosomal pH. A standard curve is obtained by equilibrating intralysosomal pH with extracellular pH using the ionophore nigericin. The protocol also includes information regarding procedures to induce lysosomal alkalinization and lysosomal membrane permeabilization.

Place, publisher, year, edition, pages
Humana Press, 2017. Vol. 1594, p. 179-189
Series
Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029 ; 1594
Keywords [en]
Bafilomycin A; Dual emission ratiometry; Flow cytometry; Lysosomal membrane permeabilization; Lysosomes; pH
National Category
Cell Biology
Identifiers
URN: urn:nbn:se:liu:diva-145059DOI: 10.1007/978-1-4939-6934-0_11PubMedID: 28456983ISBN: 978-1-4939-6932-6 (print)ISBN: 978-1-4939-6934-0 (electronic)OAI: oai:DiVA.org:liu-145059DiVA, id: diva2:1181714
Available from: 2018-02-09 Created: 2018-02-09 Last updated: 2018-02-09

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Eriksson, IdaÖllinger, KarinAppelqvist, Hanna
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Division of Cell BiologyFaculty of Medicine and Health SciencesDepartment of Clinical Pathology and Clinical GeneticsChemistryFaculty of Science & Engineering
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