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Analysis of Lysosomal pH by Flow Cytometry Using FITC-Dextran Loaded Cells
Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Diagnostikcentrum, Klinisk patologi och klinisk genetik.ORCID-id: 0000-0003-4075-159X
Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
2017 (engelsk)Inngår i: Lysosomes: Methods and Protocols / [ed] Karin Öllinger;Hanna Appelqvist, Humana Press, 2017, Vol. 1594, s. 179-189Kapittel i bok, del av antologi (Fagfellevurdert)
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.

sted, utgiver, år, opplag, sider
Humana Press, 2017. Vol. 1594, s. 179-189
Serie
Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029 ; 1594
Emneord [en]
Bafilomycin A; Dual emission ratiometry; Flow cytometry; Lysosomal membrane permeabilization; Lysosomes; pH
HSV kategori
Identifikatorer
URN: urn:nbn:se:liu:diva-145059DOI: 10.1007/978-1-4939-6934-0_11PubMedID: 28456983ISBN: 978-1-4939-6932-6 (tryckt)ISBN: 978-1-4939-6934-0 (digital)OAI: oai:DiVA.org:liu-145059DiVA, id: diva2:1181714
Tilgjengelig fra: 2018-02-09 Laget: 2018-02-09 Sist oppdatert: 2018-02-09

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