Search tips
Search criteria 


Logo of jcellbiolHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
J Cell Biol. 1985 March 1; 100(3): 965–973.
PMCID: PMC2113505

Nile red: a selective fluorescent stain for intracellular lipid droplets


We report that the dye nile red, 9-diethylamino-5H- benzo[alpha]phenoxazine-5-one, is an excellent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry. The specificity of the dye for lipid droplets was assessed on cultured aortic smooth muscle cells and on cultured peritoneal macrophages that were incubated with acetylated low density lipoprotein to induce cytoplasmic lipid overloading. Better selectivity for cytoplasmic lipid droplets was obtained when the cells were viewed for yellow-gold fluorescence (excitation, 450-500 nm; emission, greater than 528 nm) rather than red fluorescence (excitation, 515-560 nm; emission, greater than 590 nm). Nile red-stained, lipid droplet-filled macrophages exhibited greater fluorescence intensity than did nile red- stained control macrophages, and the two cell populations could be differentiated and analyzed by flow cytofluorometry. Such analyses could be performed with either yellow-gold or red fluorescence, but when few lipid droplets per cell were present, the yellow-gold fluorescence was more discriminating. Nile red exhibits properties of a near-ideal lysochrome. It is strongly fluorescent, but only in the presence of a hydrophobic environment. The dye is very soluble in the lipids it is intended to show, and it does not interact with any tissue constituent except by solution. Nile red can be applied to cells in an aqueous medium, and it does not dissolve the lipids it is supposed to reveal.

Full Text

The Full Text of this article is available as a PDF (3.7M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Goldstein JL, Brown MS. The low-density lipoprotein pathway and its relation to atherosclerosis. Annu Rev Biochem. 1977;46:897–930. [PubMed]
  • Brown MS, Goldstein JL. Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis. Annu Rev Biochem. 1983;52:223–261. [PubMed]
  • Kruth HS. Flow cytometry: rapid biochemical analysis of single cells. Anal Biochem. 1982 Sep 15;125(2):225–242. [PubMed]
  • LILLIE RD. The mechanism of Nile blue staining of lipofuscins. J Histochem Cytochem. 1956 Jul;4(4):377–381. [PubMed]
  • Dunnigan MG. The use of Nile blue sulphate in the histochemical identification of phospholipids. Stain Technol. 1968 Sep;43(5):249–256. [PubMed]
  • Chen RF. Removal of fatty acids from serum albumin by charcoal treatment. J Biol Chem. 1967 Jan 25;242(2):173–181. [PubMed]
  • HAVEL RJ, EDER HA, BRAGDON JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest. 1955 Sep;34(9):1345–1353. [PMC free article] [PubMed]
  • Goldstein JL, Ho YK, Basu SK, Brown MS. Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition. Proc Natl Acad Sci U S A. 1979 Jan;76(1):333–337. [PubMed]
  • D'Silva JB, Notari RE. Drug stability in liposomal suspensions: hydrolysis of indomethacin, cyclocytidine, and p-nitrophenyl acetate. J Pharm Sci. 1982 Dec;71(12):1394–1398. [PubMed]
  • DE DUVE C, PRESSMAN BC, GIANETTO R, WATTIAUX R, APPELMANS F. Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissue. Biochem J. 1955 Aug;60(4):604–617. [PubMed]
  • Fujiki Y, Hubbard AL, Fowler S, Lazarow PB. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol. 1982 Apr;93(1):97–102. [PMC free article] [PubMed]
  • St Clair RW, Smith BP, Wood LL. Stimulation of cholesterol esterification in rhesus monkey arterial smooth muscle cells. Circ Res. 1977 Feb;40(2):166–173. [PubMed]
  • Fowler S, Shio H, Wolinsky H. Subcellular fractionation and morphology of calf aortic smooth muscle cells. Studies on whole aorta, aortic explants, and subcultures grown under different conditions. J Cell Biol. 1977 Oct;75(1):166–184. [PMC free article] [PubMed]
  • Brown MS, Goldstein JL, Krieger M, Ho YK, Anderson RG. Reversible accumulation of cholesteryl esters in macrophages incubated with acetylated lipoproteins. J Cell Biol. 1979 Sep;82(3):597–613. [PMC free article] [PubMed]
  • Hara A, Radin NS. Lipid extraction of tissues with a low-toxicity solvent. Anal Biochem. 1978 Oct 1;90(1):420–426. [PubMed]
  • Falcone DJ, Mated N, Shio H, Minick CR, Fowler SD. Lipoprotein-heparin-fibronectin-denatured collagen complexes enhance cholesteryl ester accumulation in macrophages. J Cell Biol. 1984 Oct;99(4 Pt 1):1266–1274. [PMC free article] [PubMed]
  • Bozzo L, Vidal B de C. Nile blue sulphate and brilliant cresyl blue in fluorescence methods for the histochemical demonstration of lipids. Ann Histochim. 1968 Jul-Sep;13(3):177–183. [PubMed]
  • BERG NO. A histological study of masked lipids; stainability, distribution and functional variations. Acta Pathol Microbiol Scand Suppl. 1951;90:1–192. [PubMed]
  • McGookey DJ, Anderson RG. Morphological characterization of the cholesteryl ester cycle in cultured mouse macrophage foam cells. J Cell Biol. 1983 Oct;97(4):1156–1168. [PMC free article] [PubMed]
  • Börnig H, Geyer G. Staining of cholesterol with the fluorescent antibiotic "filipin". Acta Histochem. 1974;50(1):110–115. [PubMed]
  • Kruth HS. Filipin-positive, oil red O-negative particles in atherosclerotic lesions induced by cholesterol feeding. Lab Invest. 1984 Jan;50(1):87–93. [PubMed]
  • Muller CP, Stephany DA, Winkler DF, Hoeg JM, Demosky SJ, Jr, Wunderlich JR. Filipin as a flow microfluorometry probe for cellular cholesterol. Cytometry. 1984 Jan;5(1):42–54. [PubMed]
  • Pitas RE, Innerarity TL, Weinstein JN, Mahley RW. Acetoacetylated lipoproteins used to distinguish fibroblasts from macrophages in vitro by fluorescence microscopy. Arteriosclerosis. 1981 May-Jun;1(3):177–185. [PubMed]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press