Nile red is a fluorescent dye that effectively stains intracellular lipid droplets for detection by fluorescence microscopy and flow cytometry. It is highly specific for lipid droplets, showing greater fluorescence intensity in lipid-loaded cells compared to control cells. Nile red exhibits properties of a near-ideal lysochrome, being strongly fluorescent only in hydrophobic environments and not interacting with tissue constituents except through solution. It is soluble in lipids and does not dissolve the lipids it is intended to stain. The formation of cytoplasmic lipid droplets is a normal cellular process, consisting of neutral lipids such as triacylglycerols and cholesteryl esters. Abnormal accumulation of these droplets occurs in various pathological conditions. Flow cytometry and fluorescence microscopy are powerful techniques for analyzing cellular processes, but fluorescent dyes for lipid droplet studies under normal and pathological conditions have been lacking. Nile red was found to be an excellent stain for this purpose. Nile red is a derivative of nile blue, which was first used for histochemical detection of tissue lipids. It has been shown that nile blue and similar dyes can stain acid lipids blue and neutral lipids red. However, the fluorescence of nile red and its phenoxazone derivatives has not been widely studied. Nile red is intensely fluorescent and can serve as a sensitive vital stain for detecting cytoplasmic lipid droplets. Nile red was prepared from nile blue by dissolving it in sulfuric acid and boiling under reflux. It was then extracted into xylene and purified. Lipoproteins were isolated from human plasma and acetylated low density lipoprotein was used to induce lipid droplet formation in macrophages. Nile red staining was performed on both fixed and unfixed cells, and the results were analyzed by fluorescence microscopy and flow cytometry. Nile red staining showed intense fluorescence in lipid droplets, with greater intensity in cells with more lipid droplets. Flow cytometry confirmed the presence of lipid droplets in cells, with distinct fluorescence intensity profiles. Nile red fluorescence was more discriminative when few lipid droplets per cell were present. Nile red is a selective fluorescent stain for intracellular lipid droplets, with high specificity and sensitivity. It is a valuable tool for studying lipid droplet formation and lipid overload in cells under normal and pathological conditions.Nile red is a fluorescent dye that effectively stains intracellular lipid droplets for detection by fluorescence microscopy and flow cytometry. It is highly specific for lipid droplets, showing greater fluorescence intensity in lipid-loaded cells compared to control cells. Nile red exhibits properties of a near-ideal lysochrome, being strongly fluorescent only in hydrophobic environments and not interacting with tissue constituents except through solution. It is soluble in lipids and does not dissolve the lipids it is intended to stain. The formation of cytoplasmic lipid droplets is a normal cellular process, consisting of neutral lipids such as triacylglycerols and cholesteryl esters. Abnormal accumulation of these droplets occurs in various pathological conditions. Flow cytometry and fluorescence microscopy are powerful techniques for analyzing cellular processes, but fluorescent dyes for lipid droplet studies under normal and pathological conditions have been lacking. Nile red was found to be an excellent stain for this purpose. Nile red is a derivative of nile blue, which was first used for histochemical detection of tissue lipids. It has been shown that nile blue and similar dyes can stain acid lipids blue and neutral lipids red. However, the fluorescence of nile red and its phenoxazone derivatives has not been widely studied. Nile red is intensely fluorescent and can serve as a sensitive vital stain for detecting cytoplasmic lipid droplets. Nile red was prepared from nile blue by dissolving it in sulfuric acid and boiling under reflux. It was then extracted into xylene and purified. Lipoproteins were isolated from human plasma and acetylated low density lipoprotein was used to induce lipid droplet formation in macrophages. Nile red staining was performed on both fixed and unfixed cells, and the results were analyzed by fluorescence microscopy and flow cytometry. Nile red staining showed intense fluorescence in lipid droplets, with greater intensity in cells with more lipid droplets. Flow cytometry confirmed the presence of lipid droplets in cells, with distinct fluorescence intensity profiles. Nile red fluorescence was more discriminative when few lipid droplets per cell were present. Nile red is a selective fluorescent stain for intracellular lipid droplets, with high specificity and sensitivity. It is a valuable tool for studying lipid droplet formation and lipid overload in cells under normal and pathological conditions.