Eriksson Ida, Öllinger Karin, Appelqvist Hanna
Experimental Pathology, Department of Clinical and Experimental Medicine, Linköping University, SE-58185, Linköping, Sweden.
Division of Chemistry, Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden.
Methods Mol Biol. 2017;1594:179-189. doi: 10.1007/978-1-4939-6934-0_11.
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.
溶酶体腔的酸性环境为酸性水解酶提供了最佳环境，对于内溶酶体区室的融合/裂变以及货物分选也至关重要。有证据表明，维持溶酶体酸性对于避免疾病至关重要。在本章中，我们描述了一种使用荧光探针异硫氰酸荧光素（FITC）-葡聚糖以及适用于流式细胞术的双发射比率技术分析培养细胞中溶酶体pH值的方案。荧光标记的葡聚糖被内吞并积聚在溶酶体区室中。当在最大发射波长处分析时，FITC的荧光显示出pH依赖性变化，而在等吸收点分析时则没有变化，因此该比率可用于确定溶酶体pH值。通过使用尼日利亚菌素使溶酶体内pH与细胞外pH平衡来获得标准曲线。该方案还包括有关诱导溶酶体碱化和溶酶体膜通透性的程序的信息。
