A Simple Method to Visualize and Assess the Integrity of Lysosomal Membrane in Mammalian Cells Using a Fluorescent Dye
Fluorescent dyes have been used as “nanosensors” for visualization and determination of various processes occurring inside a cell, or intracellular events, such as cell cycle progression and intracellular trafficking. Here, we describe a novel use of acri
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Introduction The use of fluorescent dyes or enzymes with specificity to a particular target, such as a cellular receptor or an intracellular structure, are invaluable in investigating etiology of diseases and thus developing promising treatments for those diseases (1). Fluorescent dyes and enzymes have also been used to examine the mechanisms of various cellular processes such as endocytosis, exocytosis, and cell death (2). Furthermore, the morphology of intracellular structures such as the nucleus, mitochondria, lysosomes, and actin can be examined using fluorescent dyes (3). These dyes are able to fluoresce due to the presence of a functional group which will absorb energy at a specific wavelength and reemit energy at a different wavelength (4). This causes that molecule to fluoresce, enabling us to detect the presence/absence of that molecule in the experimental samples (4, 5). The amount of fluorescence can be
Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology, vol. 991, DOI 10.1007/978-1-62703-336-7_3, © Springer Science+Business Media New York 2013
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Syed K. Sohaebuddin and Liping Tang
detected and quantified through the magnitude of fluorescence. Such quantitative analysis is performed using either fluorescent plate readers or flow cytometry (6). To bolster the quantitative data, qualitative assessment can be performed which allows us to observe the physical location(s) of our target in the experimental sample as well as quantify their presence at a particular location(s) in the same sample. The use of both quantitative and qualitative analysis leads to lucid understanding of experimental results and observed phenomenon. There are, however, some dyes which are not compatible for qualitative assessment mainly because irradiation of the dyes to certain wavelengths of light causes damage to the cell’s internal organelles. Damaged organelles may leak the dye to other cellular components such as the cytoplasm, leading to false qualitative results (7). Acridine orange is one of these dyes which, when irradiated with intense blue light, causes damage to the lysosome’s membrane (8). Acridine orange is a weak base metachromatic dye capable of crossing plasma membranes and staining nucleic acids and lysosomes. At low concentrations, it can differentiate lysosomes (reddish-orange granules) from other cellular components (diffuse green) (9). Acridine orange molecules become protonated under acidic conditions and hence get trapped within lysosomes. Accumulation of acridine orange molecules in lysosomes leads to a shift in excitation from green = 530 nm to red = 620 nm (10). When pH of the lysosomes rises or if their membranes are damaged, acridine orange molecules become deprotonated, and these molecules can then cross back into the cytoplasm. This shifts the emission back from red to green (11). Therefore, extended exposure (>1 min) of acridine orange loaded cells to blue light leads to lysosomal membrane damage and a shift in the lysosome
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