Nile red staining of lysosomal phospholipid inclusions

Abstract

We have employed the fluorescent dye nile red to distinguish between normal cells and cells containing lysosomal accumulations of phospholipids. When fibroblasts from an individual with a genetic deficiency in lysosomal sphingomyelinase activity (Niemann-Pick disease) were stained with nile red and visualized by fluorescence microscopy, orange-colored inclusions were observed throughout the cytoplasm. The orange fluorescent bodies could be distinguished from the neutral lipid droplets that fluoresce a brilliant yellow-gold in the presence of nile red. These inclusions were also observed in alveolar macrophages obtained from rats treated with amiodarone, an antiarrhythmic agent known to produce lysosomal phospholipidosis. Flow cytofluorometric analysis revealed that staining of these phospholipid-rich macrophages with nile red can distinguish them from control alveolar macrophages. These results demonstrate that nile red can be employed for the rapid staining of cellular phospholipid inclusions.

Labeling nanoparticles: Dye leakage and altered cellular uptake

“…Microscopy is another method used for studying uptake and distribution of NPs (18,22,37,43). However, microscopy should be employed with care in the assessment of dye leakage, as free hydrophobic dyes will bind to intracellular hydrophobic molecules resulting in both diffuse and spotted staining pattern (12,24,30,33), thereby making the dye hard to separate from the fluorescence of intact NPs. In a previous study, we have shown that PBCA-NPs with NR668 are taken up in PC3 cells by endocytosis, verified by the use of time consuming intracellular spectral microscopy and fluorescence-lifetime imaging microscopy (FLIM) (12).…”

“…Various procedures have been developed to evaluate NP-labeling stability (18,20,23,(25)(26)(27)(28)(29). However, the majority of these assays do not include cells or serum, which could strongly affect dye release as these serve as acceptor compartments for released dye in vivo (18,24,(29)(30)(31)(32)(33).…”

7‐Ketocholesterol favors lipid accumulation and colocalizes with Nile Red positive cytoplasmic structures formed during 7‐ketocholesterol–induced apoptosis: Analysis by flow cytometry, FRET biphoton spectral imaging microscopy, and subcellular fractionation

“…These images were analyzed by the Factor Analysis of Medical Image Sequences (FAMIS) algorithm, which provides factor curves and images (31,32). The ability to colocalize 7KC (emitting a blue fluorescence under ultraviolet [UV] light) (33) and cellular lipids stained with NR is possible because of the overlap of the emission spectra of 7KC with the excitation spectra of NR (33)(34)(35). In addition, subcellular fractionation associated with gas chromatography and mass spectrometry was used to characterize NR-positive cytoplasmic structures.…”

A Novel Technique For Mapping the Lipid Composition of Atherosclerotic Fatty Streaks by En Face Fluorescence Microscopy

“…Fluorescence and light microscopy have been instrumental in locating and identifying lipid components of atherosclerotic lesions in animal models and human biopsy tissues (Brown et al 1992;Seifert 1989;Lupu et al 1987;Fowler and Greenspan 1985;Kruth 1984aKruth ,b,1985Kruth and Fry 1984). Immunohistochemical (IH) methods have been used to distinguish populations of smooth muscle cells and macrophages in atherosclerotic lesions (Masuda and Ross 1990;Rosenfeld et al 1987;Tsukada et al 1986), and many authors have used electron microscopy to characterize cell types and investigate intracellular aspects of atherosclerosis (Masuda and Ross 1990;Lupu et al 1987;Rosenfeld et al 1987).…”