| Chapter title |
Analysis of Lysosomal pH by Flow Cytometry Using FITC-Dextran Loaded Cells
|
|---|---|
| Chapter number | 11 |
| Book title |
Lysosomes
|
| Published in |
Methods in molecular biology, April 2017
|
| DOI | 10.1007/978-1-4939-6934-0_11 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-6932-6, 978-1-4939-6934-0
|
| Authors |
Ida Eriksson, Karin Öllinger, Hanna Appelqvist |
| Editors |
Karin Öllinger, Hanna Appelqvist |
| 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. |
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Demographic breakdown
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|---|---|---|
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| Other | 0 | 0% |
| Unknown | 16 | 52% |