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  3. Analysis of Lysosomal pH by Flow Cytometry Using FITC-Dextran Loaded Cells

Analysis of Lysosomal pH by Flow Cytometry Using FITC-Dextran Loaded Cells

  • Methods Mol Biol. 2017;1594:179-189. doi: 10.1007/978-1-4939-6934-0_11.
Ida Eriksson 1,  Karin Öllinger 2,  Hanna Appelqvist 3
Affiliations

Affiliations

  • 1 Experimental Pathology, Department of Clinical and Experimental Medicine, Linköping University, SE-58185, Linköping, Sweden.
  • 2 Experimental Pathology, Department of Clinical and Experimental Medicine, Linköping University, SE-58185, Linköping, Sweden. [email protected].
  • 3 Division of Chemistry, Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden.
PMID: 28456983 DOI: 10.1007/978-1-4939-6934-0_11
Abstract

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.

Keywords

Bafilomycin A; Dual emission ratiometry; Flow cytometry; Lysosomal membrane permeabilization; Lysosomes; pH.

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